MEDC0066

Practical Laboratory Research Skills
实用实验室研究技能

(PLRS)

Protocol Manual 2024-25
协议手册 2024-25

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姓名：...................................................................

Programme: ………………………………………………….
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TIMETABLE
时间表

Sessions will be taught by Prof J Norman, Dr J Donovan and Dr M Plate and PGTAs unless otherwise indicated.
除非另有说明，否则课程将由 J Norman 教授、J Donovan 博士、M Plate 博士和 PGTA 教授讲授。

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协议检查页码！

APPENDICES
附录

INTRODUCTION
引言

LABORATORY SAFETY
实验室安全

• Health and safety are everyone’s responsibility, and you need to make sure you know about the potential hazards of any activity that you plan to do in a laboratory.
- 健康和安全是每个人的责任，您需要确保了解您计划在实验室进行的任何活动的潜在危险。

• UCL has in place, policies to ensure that staff, students and visitors are safe in their workplace, and these are in line with the Health and Safety at Work Act 1974 (HASAWA).
- UCL 制定了相关政策，以确保员工、学生和访客在工作场所的安全，这些政策符合 1974 年《工作场所健康与安全法》（HASAWA）。

GENERAL SAFETY MEASURES
一般安全措施

NO eating or drinking in the laboratory (including chewing gum and drinking water!).
禁止在实验室内吃喝（包括嚼口香糖和喝水！）。

Make sure you know where to dispose of waste materials (see Waste Management, below).
确保知道在哪里处理废料（见下文的废物管理）。

Wash your hands before leaving the laboratory.
离开实验室前要洗手。

PERSONAL PROTECTIVE EQUIPMENT (PPE)
个人防护设备（PPE）

Lab coats
实验服

Lab coats must be worn in any wet laboratory. Lab coats must be high neck coats with knitted cuffs and press stud fastenings (Howie style) and must be fastened properly.
在任何潮湿的实验室都必须穿实验服。实验服必须是高领大衣，袖口有针织物和按扣（Howie 式），且必须系好。

All jewelry, scarves, ties, accessories etc that might become contaminated or cause entanglement should be contained within the lab coat.
所有可能被污染或造成缠绕的首饰、围巾、领带、配饰等都应放在白大褂内。

Long hair should be tied-up.
长发应扎起来。

Safety glasses
安全眼镜

Eye protection must be worn in the laboratory, even if you wear glasses for vision correction.
在实验室内必须佩戴护目镜，即使您佩戴的是矫正视力的眼镜。

Suitable clothing
合适的服装

Although footwear and appropriate clothing are not considered PPE, they must provide minimum protection for the skin and feet.
虽然鞋类和适当的服装不属于个人防护设备，但它们必须为皮肤和脚部提供最起码的保护。

Footwear should be fully enclosed and cover the feet and must be able to resist hazardous substances or at least slow the exposure of the feet to hazardous substances.
鞋袜应完全封闭并覆盖双脚，必须能够抵御危险物质或至少减缓双脚接触危险物质的速度。

Clothing should cover the legs to reduce skin exposure to splashes and spills.
衣服应遮住腿部，以减少皮肤接触飞溅物和溢出物的机会。

Gloves
手套

Where appropriate, wear gloves.
在适当的情况下，戴上手套。

Note: Gloves must be removed before touching e.g. door handles, lift call buttons or YOUR PHONE
注意：在触摸门把手、电梯呼叫按钮或您的手机等设备之前，必须脱下手套。

WASTE MANAGEMENT
废物管理

Waste generated in a laboratory may be one of the following:
实验室产生的废物可能是以下废物之一：

Non-hazardous domestic waste (e.g. paper, packaging) – Clear or black bags
无害生活垃圾（如纸张、包装） - 透明或黑色袋子

Clinical or biological waste (e.g. clinical samples, cell culture vessels gloves) – Yellow bags, Tiger bags (striped) or autoclave waste bags
临床或生物废物（如临床样本、细胞培养器皿手套） - 黄色袋、虎纹袋（条纹）或高压灭菌废物袋

Sharps (e.g. needles, scalpel blades, broken glass) – Yellow plastic sharps bins
利器（如针头、手术刀刀片、碎玻璃） - 黄色塑料利器箱

Pipets and pipet tips – Yellow and green bio-bins (bench or floor standing)
移液器和吸头 - 黄色和绿色生物箱（台式或落地式）

Other: Ask the lab manager or the Health and Safety Officer.
其他：请咨询实验室经理或健康与安全官。

ACCIDENTS
事故

Accidents do happen in laboratories.
实验室确实会发生意外。

Accidents can be due to human error, faulty equipment or inadequate training.
事故的原因可能是人为失误、设备故障或培训不足。

ALL accidents should be reported to a member of staff.
所有事故都应向工作人员报告。

RISK ASSESSMENTS
风险评估

To reduce the risk of damage/injury a Risk Assessment should be conducted before beginning any activity. A Risk Assessment is a structured process intended to help identify hazards associated with activities in the workplace, evaluate the potentially harmful consequences of the hazards, and find ways to avoid or minimize the hazard. The process ultimately gives rise to a written Safe System of Working which aims to ensure that risks are reduced to levels which are as low as reasonably practicable.
为降低损害/伤害风险，在开始任何活动之前都应进行风险评估。风险评估是一个结构化的过程，旨在帮助识别与工作场所活动有关的危险，评估危险可能造成的有害后果，并找到避免或最大限度减少危险的方法。这一过程最终会形成书面的安全工作制度，其目的是确保将风险降低到合理可行的最低水平。

Definitions:
定义

• Hazard: A property of a substance, activity or situation that has the potential to cause harm.
- 危害：可能造成伤害的物质、活动或情况的属性。

• Risk: The probability that the harm linked to a hazard will actually occur, taking into account also the severity of that harm.
- 风险：与危害相关的伤害实际发生的概率，同时考虑到伤害的严重程度。

HEALTH AND SAFETY IN PRACTICE
健康与安全实践

Read the following scenarios and make a note of any risks associated with the activities.
阅读以下情景，并记下与活动相关的任何风险。

Scenario 1: A PhD student has recently started working in a research laboratory. They make pasta at home one evening and take some of the leftover pasta to work for lunch the next day. There is a fridge in the laboratory so they put their lunch in the fridge to keep it cool. For their project the student is analyzing samples from patients with liver disease. They collect a human liver sample and divide the sample in two: They freeze one half of the sample in liquid nitrogen (−196°C) for future analysis and use the other half for acid digestion. Acid digestion involves incubating the sample in concentrated hydrochloric acid (12M HCl) for hours. After finishing the experiment, the student disposes of their gloves in the recycling bin, washes their hands, collects their lunch-box from the fridge and goes home.
情景 1：一名博士生最近开始在研究实验室工作。一天晚上，他们在家里做意大利面，第二天中午带着一些剩面去上班。实验室里有一台冰箱，所以他们把午餐放在冰箱里保持凉爽。学生的项目是分析肝病患者的样本。他们收集人体肝脏样本，并将样本一分为二：他们将一半样本冷冻在液氮中（-196°C）以备将来分析，另一半样本用于酸解。酸解包括将样本在浓盐酸（12M HCl）中培养数小时。实验结束后，学生将手套扔进回收箱，洗净双手，从冰箱中取出饭盒，然后回家。

Scenario 2: A student has joined a research laboratory for their project. The experiments are interesting but time-consuming, they start at 9.00 but don’t finish until 22:30 and the student has to stay in the laboratory alone to make sure all the extraction columns are working during the experiment. The student cannot work without coffee, so they get a coffee from Starbucks and drink it in the laboratory while doing the experiments. The student is using a technique called a radioimmunoassay which involves using insulin tagged with a radioactive label to measure insulin levels in blood samples from obese mice. After finishing the experiments, the student disposes of their gloves in a yellow waste bag and goes home.
情景 2：一名学生参加了一个研究实验室的项目。实验很有趣，但很耗时，9:00 开始，22:30 才结束，学生必须一个人待在实验室里，确保实验过程中所有萃取柱都在工作。学生的工作离不开咖啡，因此他们从星巴克买了一杯咖啡，在实验室里边喝咖啡边做实验。学生正在使用一种叫做放射免疫分析的技术，即使用带有放射性标签的胰岛素来测量肥胖小鼠血液样本中的胰岛素水平。实验结束后，学生将手套放入黄色垃圾袋中，然后回家。

ESSENTIAL LABORATORY SKILLS
基本实验技能

1. SOLUTIONS
1.解决方案

Making solutions is one of the most common activities in the lab. Being able to make complex solutions quickly and accurately is an essential skill.
配制溶液是实验室中最常见的工作之一。能够快速准确地配制复杂的溶液是一项基本技能。

Work through the following problems (please show ALL your working/calculations in your lab book):
完成下列问题（请在实验本上展示你的所有作业/计算）：

You need to make a solution of 1M sodium chloride (NaCl).
您需要配制 1M 的氯化钠（NaCl）溶液。

What information do you need to know to make the solution?
制定解决方案需要了解哪些信息？

b) You need to make 50ml of a solution with a final concentration: 0.2% ethanol, 5mM EDTA, 20mM NaCl. The tube already contains 5ml 2% (v/v) ethanol and 5ml 50mM EDTA in water.
b) 你需要配制 50 毫升最终浓度为 0.2%乙醇、5 毫摩尔乙二胺四乙酸、20 毫摩尔氯化钠的溶液：0.2%乙醇、5mM EDTA、20mM NaCl。试管中已含有 5 毫升 2%（体积分数）乙醇和 5 毫升 50mM EDTA 水溶液。

You have a stock solution of 0.5M NaCl.
您有一份 0.5M NaCl 的储备溶液。

What volume of 0.5M NaCl you need to add to the tube?
您需要在试管中加入多少体积的 0.5M NaCl？

What volume of water do you need to complete the solution?
完成溶液需要多少体积的水？

c) You have stock solutions of:
c) 您有以下物质的库存溶液：

10% Sodium dodecyl sulphate (SDS)
10% 十二烷基硫酸钠 (SDS)

0.5M EDTA

1.5M Tris

Water
水

Calculate the volumes of each solution you would need to make 50mls of solution containing a final concentration of 0.2% SDS, 0.1M EDTA, 0.3M Tris.
计算配制 50 毫升最终浓度为 0.2%SDS、0.1M EDTA 和 0.3M Tris 的溶液所需的每种溶液的体积。

d) You have 1ml of a 1M solution NaCl. You need to make 10ml of 0.1mM NaCl. How do you perform the dilution? Write out all the steps.
d) 您有 1 毫升 1M NaC 溶液。您需要配制 10 毫升 0.1 毫摩尔 NaCl 溶液。如何进行稀释？写出所有步骤。

e) Tube A contains a dye called Trypan Blue.
e) 试管 A 中含有一种叫做胰蓝的染料。

You need to dilute this by 10,000x to give 1ml of the final solution.
您需要将其稀释 10 000 倍，才能得到 1 毫升的最终溶液。

Write out all the steps to perform a serial dilution with water and make 1ml of the final solution.
写出用水进行系列稀释并配制 1 毫升最终溶液的所有步骤。

2. BASIC LAB EQUIPMENT
2.基本实验室设备

Each apparatus will be demonstrated by a member of staff.
每种器械都将由一名工作人员进行演示。

i) Pipettors and pipettes.
i) 移液器和移液管。

Pipette the following volumes of water:
移取以下体积的水：

You have available: 0.5ml, 1.5ml, 15ml and 50ml tubes and 5,10 and 25ml pipettes. Select the pipettes that allow you to measure the required volume most accurately and individual tubes that can accommodate the volume you are measuring but are not unnecessarily large.
您可以获得0.5 毫升、1.5 毫升、15 毫升和 50 毫升试管以及 5 毫升、10 毫升和 25 毫升移液管。选择能让您最准确地测量所需体积的移液管，以及能容纳您要测量的体积但又不会太大的试管。

Pipette the following volumes:
移取以下体积：

1.2ml, 2.5ml, 5ml
1.2毫升、2.5毫升、5毫升

3.3ml, 5.4ml, 7.5 ml
3.3 毫升、5.4 毫升、7.5 毫升

11ml, 17ml, 21ml
11 毫升、17 毫升、21 毫升

Compare your tubes to the demonstration tubes. Are the volumes the same?
将您的试管与演示试管进行比较。体积是否相同？

Use the appropriate Gilson pipette and tips to pipette the following volumes into individual 1.5ml microfuge tubes:
使用合适的 Gilson 移液器和吸头，将以下体积移入 1.5 毫升的微量离心管中：

P1000 (blue tips) – 1000ml, 250ml, 750m
P1000（蓝色尖端）- 1000 米、250 米、750 米l

P200 (yellow tips) – 130ml, 40ml, 25m
P200（黄色喷嘴）- 130毫升、40米、25米l

P20 (yellow tips) – 19ml, 10ml, 3m
P20（黄色喷嘴）- 19毫升、10米、3米l

Compare your tubes to the demonstration tube. Are the volumes the same?
将您的试管与示范试管进行比较。体积是否相同？

ii) Balance
ii) 余额

Task weigh a known volume of NaCl:
任务称量已知体积的氯化钠：

Select a weighboat
选择地磅

Zero (Tare) the balance
天平清零（去皮

Weigh out 750mg NaC
称出 750 毫克 NaCl

Make sure the balance is clean when you have finished.
完成后，确保天平是干净的。

iii) pH meter
iii) pH 计

Making buffers requires a rapid and accurate way of measuring the pH of a solution. A pH meter is a device used for measuring the pH of solutions. A typical pH meter consists of a special measuring electrode (with a glass bulb) which is attached to an electronic meter that measures and displays the pH reading.
制作缓冲液需要快速准确地测量溶液的 pH 值。pH 计是一种用于测量溶液 pH 值的设备。典型的 pH 计由一个特殊的测量电极（带玻璃球）组成，电极连接到一个电子表上，电子表测量并显示 pH 读数。

Note: The glass bulb of the electrode should always be kept wet.
注意：电极的玻璃球应始终保持湿润。

Task measure to pH of two different solutions:
测量两种不同溶液的 pH 值：

Calibrate the pH meter
校准 pH 计

Measure the pH of Solution X
测量溶液 X 的 pH 值

Measure the pH of Solution Y
测量溶液 Y 的 pH 值

Make a note of the pH of both solutions.
记下两种溶液的 pH 值。

iv) Centrifuge
iv) 离心机

Centrifugation is the mechanical process of separating mixtures by applying centrifugal forces. It is one of the most widely used methods in the laboratory. In common protocols a mixture is separated into two fractions – a pellet at the bottom of the tube containing material that sediments at the applied centrifugal force and a liquid supernatant containing materials that did not sediment.
离心是通过离心力分离混合物的机械过程。它是实验室中使用最广泛的方法之一。在常见的方案中，混合物会被分离成两个部分--试管底部的颗粒（包含在离心力作用下沉淀的物质）和液体上清液（包含未沉淀的物质）。

A centrifuge has two main elements: a rotor to hold the samples to be centrifuged and a motor to generate the force. The rotor sits in chamber to contain the damage in the event of a failure during centrifugation.
离心机有两个主要部件：一个是用于盛放待离心样品的转子，另一个是用于产生离心力的电机。转子安装在腔体内，以便在离心过程中发生故障时防止损坏。

Units used in centrifugation:
用于离心的单位：

• RPM (Revolutions per minute): RPM is simply the speed of the motor.
- RPM（每分钟转数）：RPM 就是电机的转速。

• RCF (Relative centrifugal force): RCF is the gravitational force that is generated by the centrifuge. The rate of sedimentation of particles depends on the acceleration of the rotor. RCF is often expressed relative to the acceleration of gravity (g). For example, 1000 x g means centrifugation force 1000 times the acceleration of gravity.
- RCF（相对离心力）：RCF 是离心机产生的重力。颗粒的沉降速度取决于转子的加速度。RCF 通常相对于重力加速度 (g) 来表示。例如，1000 x g 表示离心力是重力加速度的 1000 倍。

RCF depends on both the speed (RPM) as well as the radius of the rotor. RCF can be determined either by a formula:
RCF 既取决于转速（RPM），也取决于转子的半径。RCF 可以通过公式确定：

RCF = 1.12 x R x (RPM/1000)² (R is the radius of the rotor measured in millimeters).
RCF = 1.12 x R x (RPM/1000)²（R 是转子的半径，单位为毫米）。

Note: If the radius of the rotor is not known, it will need to be measured. The radius is measured from the centre of the rotor to the point that would be the bottom of the sample tube.
注：如果不知道转子的半径，则需要进行测量。半径是从转子中心到样品管底部的点测量的。

Or, by using a nomogram (overleaf) to find the RCF that is generated for a given RPM.
或者使用额定值图（见左页）找出给定转速下产生的 RCF。

Use a) the formula and b) the nomogram to calculate the rotor speed to produce a RCF of 1500 x g
使用 a) 公式和 b) 名义图计算转子转速，以产生 1500 x g 的 RCF。

Note: When describing centrifugation conditions e.g. in the methods section of a paper or dissertation, it is best to use RCF as this is constant whereas the RPM needed to generate a specific RCF varies depending on the centrifuge and rotor that are being used.
注：在描述离心条件时，例如在论文或学位论文的方法部分，最好使用 RCF，因为 RCF 是恒定的，而产生特定 RCF 所需的 RPM 会因使用的离心机和转子而异。

Nomogram to estimate the speed in revolutions per minute (RPM) needed to produce a specific relative centrifugal force (RCF).
用于估算产生特定相对离心力 (RCF) 所需的转速（每分钟转数）的示意图。

Set up the centrifuge so it is balanced (tubes of equal weight diagonally opposite each other in the rotor).
设置离心机使其平衡（转子中重量相等的试管斜对面）。

Calculate the rotor speed (RPM) to produce 1500 x g RCF
计算产生 1500 x g RCF 的转子转速（RPM）。

Program the centrifuge to spin for 2 mins at 1500 x g
将离心机设置为以 1500 x g 的转速旋转 2 分钟

At the end of the session make sure your bench is tidy and all waste has been disposed of in the correct waste containers. Ensuring you have a tidy bench is an essential lab skill!
课程结束时，请确保您的工作台整洁，所有废弃物都已放入正确的废弃物容器中。确保工作台整洁是一项基本的实验技能！

You must complete the tasks and have them signed off before leaving the class.
您必须完成任务并签字确认后才能离开课堂。

H&E is the most commonly used staining procedure in histology and involves two dyes, haematoxylin which stains cell nuclei a deep blue-purple colour and eosin which stains the cytoplasm, extracellular matrix and other proteinaceous structures pink. H&E provides a comprehensive picture of the microanatomy of cells, tissues and organs. It can be used to stain paraffin-embedded tissue sections, frozen sections and cell smears.
H&E 是组织学中最常用的染色方法，包括两种染料，一种是将细胞核染成深蓝紫色的血色素，另一种是将细胞质、细胞外基质和其他蛋白质结构染成粉红色的伊红。H&E 能全面反映细胞、组织和器官的微观解剖结构。它可用于对石蜡包埋的组织切片、冷冻切片和细胞涂片进行染色。

The staining procedure for paraffin-embedded tissue sections follows a basic protocol:
石蜡包埋组织切片的染色程序遵循基本方案：

Dewaxing
脱蜡

Rehydration
补液

Haematoxylin
血色素

Differentiation
差异化

Bluing
蓝化

Eosin
曙红

Dehydration
脱水

Clearing
清除

Cover-slipping
封面滑动

Equipment
设备

Coplin jars
科普林罐子

Pencil
铅笔

Staining trays
染色盘

Forceps
镊子

Pastettes
粉饼

Wash bottle
洗涤瓶

Liquid waste container
液体废物容器

Slides with tissue sections
带有组织切片的载玻片

Coverslips
盖玻片

Timer
计时器

Paper towels
纸巾

Light microscope
光学显微镜

Sections have been deparaffinised by immersing slides in 3 changes of Histoclear or Xylene (5 min, 2 min, 1 min each) and rehydrated by immersing slides in graded ethanols: 100% (3 min), 95% (2 min) and 70% (2 min) followed by water.
将切片浸入 Histoclear 或二甲苯中 3 次（每次 5 分钟、2 分钟、1 分钟）进行脱石蜡处理，然后将切片浸入不同浓度的乙醇：100%（3 分钟）、95%（2 分钟）和 70%（2 分钟），再用水冲洗。

Collect slides (2 slides labelled A and B) from the fume hood in a Coplin jar containing water.  
从通风橱中收集幻灯片（2 张幻灯片，分别标为 A 和 B），放入装有水的 Coplin 瓶中。

Rinse once with deionized water to remove residual ethanol. 
用去离子水冲洗一次，去除残留的乙醇。

Drain the slides well before staining (touch the corner of the slide to a paper towel to draw excess water off).
染色前将玻片充分沥干（用纸巾接触玻片一角，吸干多余水分）。

Label slides with your initials using a pencil and writing in the white/frosted area at the end of the slide.
用铅笔在幻灯片末端的白色/磨砂区域写上姓名缩写，并在幻灯片上贴上标签。

Place slides with the sections facing upwards on the rack in a staining tray, apply enough filtered haematoxylin (H) to completely cover the section and incubate for 5 min. 
将切片朝上放在染色盘的架子上，涂上足够的过滤血色素（H）以完全覆盖切片，然后孵育 5 分钟。

Wash with water and drain.  
用清水洗净后沥干。

Apply enough Bluing Reagent (BR) to completely cover the section and incubate for 20 seconds to differentiate the stain (until the haematoxylin only stains cell nuclei). 
涂抹足够的蓝化试剂（BR），使其完全覆盖切片，然后孵育 20 秒钟，使染色剂分化（直到血色素只染色细胞核）。

Wash with water and drain as above.
按上述方法用水清洗并沥干。

Apply enough eosin (E) to completely cover the section and incubate for 4 min.
涂上足够的伊红（E），使其完全覆盖切片并孵育 4 分钟。

Wash away the stain with water and drain.  
用水洗净污渍并沥干。

Dehydrate sections by incubation in graded ethanols: 70% (1 min), 95% (1 min), 100% (2 min). 
将切片放入不同浓度的乙醇中脱水：70%（1 分钟）、95%（1 分钟）、100%（2 分钟）。

In the fume hood, incubate the sections in 3 changes of xylene (1 min, 1 min, 2 min each). 
在通风橱中，将切片在二甲苯中培养 3 次（每次 1 分钟、1 分钟、2 分钟）。

To mount the sections using a coverslip – place a drop of mountant at the edge of the slide and gently lower the coverslip to spread the mountant over the section, take care to avoid bubbles. Allow to dry.
使用盖玻片镶嵌切片--在载玻片边缘滴一滴镶嵌剂，然后轻轻放下盖玻片，使镶嵌剂铺满切片，注意避免产生气泡。待其干燥。

Observe the slides on a light microscope
在光学显微镜上观察玻片

TIPS ON USING THE MICROSCOPE:
使用显微镜的技巧：

• Always use the lowest power/magnification to find the focus and then work your way up to higher power.
- 始终使用最低的功率/放大倍率找到焦点，然后再逐步提高功率。

• Start with the coarse focus and once you can see the image, refine with the fine focus knob. Once one slide is in focus, you may find that you can swap slides without having to start all over again.
- 从粗对焦开始，一旦能看到图像，就用细对焦旋钮细化。一旦一张幻灯片对好焦，您可能会发现您可以交换幻灯片，而无需重新开始。

• Although it is tempting to look down just one eye-piece and focusing with both eyes is tricky at first, try to use both eyepieces (it may help to adjust the distance between the eye-pieces).
- 虽然只向下看一个目镜很诱人，而且开始时双眼对焦也很困难，但还是要尽量使用两个目镜（调整目镜之间的距离可能会有帮助）。

For each slide, answer the following questions and note your answers in your lab book:
针对每张幻灯片，回答下列问题，并将答案记在实验本上：

Has the staining worked ie. purple nuclei, pink cytoplasm?
染色是否有效，即紫色的细胞核，粉红色的细胞质？

Is there clear distinction between the two stains?
这两种污渍有明显区别吗？

What tissue do you think it is?
你认为这是什么组织？

What structures can you see?
你能看到什么结构？

What types of cells are present?
有哪些类型的细胞？

What differences are observed between the two slides?
两张幻灯片有什么不同？

Image analysis
图像分析

There is a wide range of software available for analysis of scientific images depending on the microscope you are using and the type of analysis you wish to perform. One very widely-used, publicly available software is ImageJ:
根据您使用的显微镜和希望进行的分析类型，有多种软件可用于分析科学图像。其中一个使用非常广泛的公开软件是 ImageJ：

https://imagej.net/software/imagej2

Protocol 2: Introduction to tissue culture, thawing cells from cryogenic stocks
规程 2：组织培养入门，解冻低温储存的细胞

Cell culture refers to the removal of cells from an organism and their maintenance/growth in a favourable artificial environment that aims to retain the physiological and biochemical characteristics of the original tissue. It is important that we attempt to mimic the physiological conditions for a given cell type. Cell culture is a widely used technique and provides model systems for:
细胞培养是指将细胞从生物体中取出，并在有利的人工环境中维持/生长，目的是保留原始组织的生理和生化特征。我们必须尝试模拟特定细胞类型的生理条件。细胞培养是一种广泛使用的技术，它为以下方面提供了模型系统：

Studying the normal/abnormal physiology and biochemistry of cells
研究细胞的正常/非正常生理学和生物化学

Modelling disease
疾病模型

Effects of drugs and toxic compounds on the cells
药物和有毒化合物对细胞的影响

Drug screening and development
药物筛选和开发

Large scale manufacturing of biological compounds (e.g. vaccines, therapeutic proteins)
大规模生产生物化合物（如疫苗、治疗性蛋白质等）

Cultured cells require a sterile environment and a supply of nutrients for growth. The environment should also be stable in terms of pH and temperature. Many media have been developed and are produced commercially. Media contain inorganic salts, carbohydrates, amino acids, vitamins, fatty acids and lipids, proteins and peptides and may also contain specific factors to promote growth of particular cell types. Where all constituents are known the medium is referred to as a defined medium. Many media contain serum (most often foetal bovine serum) which is a complex and variable mixture of factors and enhances cell proliferation. Cell conditions vary for each cell type, failure to provide appropriate conditions can slow growth or even result in cell death.
培养细胞的生长需要无菌环境和营养供应。环境的酸碱度和温度也应稳定。目前已开发出许多培养基，并已投入商业生产。培养基含有无机盐、碳水化合物、氨基酸、维生素、脂肪酸和脂质、蛋白质和肽，还可能含有促进特定类型细胞生长的特定因子。已知所有成分的培养基被称为定义培养基。许多培养基都含有血清（最常见的是胎牛血清），这是一种复杂多变的混合因子，可促进细胞增殖。每种细胞类型的细胞条件各不相同，如果不能提供适当的条件，就会减缓生长速度，甚至导致细胞死亡。

Example of a commonly used cell culture medium:
常用细胞培养基示例：

Dulbecco’s modified Eagle’s Medium (DMEM) contains Na⁺, K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Cl^- , SO₄^2- , PO₄^3-, glucose, 20 amino acids, 10 vitamins, inositol, glutathione and Phenol red (as a pH indicator). Foetal calf serum (FCS, sometimes also referred to as foetal bovine serum (FBS) is usually added at 10-20% v/v to provide a source of growth factors and nutrients. Antibiotics (usually penicillin and streptomycin used at a final concentration of 100U/ml and 0.1mg/ml, respectively) are also added and sometimes an antimycotic (often amphotericin B, final concentration 0.25mg/ml). Antibiotic/antimycotic mixture can be purchased commercially e.g. from Sigma, as a 100X stock.
杜氏改良老鹰培养基（DMEM）含有 Na ⁺ 、K ⁺ 、Ca ^{2 +} 、Fe ²⁺ 、Mg ²⁺ 、Cl ^- 、SO ₄ ^{2 -} 、 PO ₄ ^3- 、葡萄糖、20 种氨基酸、10 种维生素、肌醇、谷胱甘肽和酚红（作为 pH 指示剂）。通常添加 10-20% v/v 的胎牛血清（FCS，有时也称为胎牛血清（FBS）），以提供生长因子和营养物质。此外，还加入抗生素（通常为青霉素和链霉素，最终浓度分别为 100U/ml 和 0.1mg/ml），有时还加入抗霉菌剂（通常为两性霉素 B，最终浓度为 0.25mg/ml）。抗生素/抗真菌剂混合物可从市场上购买，如从 Sigma 公司购买 100X 的储存液。

Equipment
设备

Cell Culture Laboratory
细胞培养实验室

Culture vessels and consumables: Sterile disposable tissue culture plasticware – culture vessels, pipettes and tubes. A range of vessels are available for growing cells but the most commonly used are flasks, Petri dishes and multi-well plates. The base material of vessels, polystyrol, alone is not sufficient for adhesion of most cells and the surface is modified to make it more charged. All suppliers of cell culture plasticware treat the surfaces of tissue culture vessels to produce surface modifications.
培养皿和耗材：一次性无菌组织培养塑料器皿--培养皿、移液管和试管。用于培养细胞的器皿种类繁多，但最常用的是烧瓶、培养皿和多孔板。培养皿的基本材料聚苯乙烯本身不足以粘附大多数细胞，因此需要对其表面进行改性，使其更具电荷性。所有细胞培养塑料器皿供应商都会对组织培养器皿的表面进行处理，使其表面改性。

Laminar flow cabinet (also referred to as a Tissue culture hood or a Class II hood) to provide a sterile working environment. Note: All cell culture work is done using aseptic technique.
层流柜（也称为组织培养罩或二级罩），提供无菌工作环境。注：所有细胞培养工作均采用无菌技术进行。

Incubator (37^oC, 5-10% CO₂) providing optimal conditions for cell growth
培养箱（37 ^o C，5-10% CO ₂ ）为细胞生长提供最佳条件

Waterbath (37^oC) for pre-warming solutions
用于预热溶液的水浴槽（37 ^o C）

Inverted light microscope
倒置光学显微镜

Centrifuge for pelleting cells
用于细胞造粒的离心机

Tips for aseptic technique and working the hood
无菌技术和工作罩小贴士

Use standard personal protective equipment (PPE) - lab coat, gloves and eye protection, make sure long hair is tied back or covered.
使用标准的个人防护设备（PPE）--白大褂、手套和护目镜，确保长发束起或盖住。

Make sure all the surface is free of clutter.
确保所有表面没有杂物。

Clean the working area inside the hood with 70% ethanol (EtOH)/IMS.
用 70% 的乙醇 (EtOH)/IMS 清洁罩内的工作区。

Before transferring bottles into the hood, disinfect all bottles with 70% EtOH/IMS by spraying or wiping.
在将瓶子转移到罩内之前，用 70% EtOH/IMS 喷洒或擦拭所有瓶子进行消毒。

In general, reagents and bottles are usually kept on the left-hand side and the consumables and discard beakers to the right-hand side of the workstation, keeping a central area clear for working (everyone finds the best set up for them).
一般来说，试剂和瓶子通常放在工作站的左侧，消耗品和废弃烧杯放在工作站的右侧，保持中央区域的工作空间（每个人都能找到最适合自己的设置）。

Change or clean gloves with 70% EtOH/IMS if they are taken out of the hood or you suspect they might be contaminated.
如果手套从罩中取出或怀疑手套可能受到污染，请更换手套或用 70% EtOH/IMS 清洗手套。

Make sure any spills are immediately wiped up with a tissue and the affected area wiped with 70% EtOH/IMS.
确保立即用纸巾擦掉任何溢出物，并用 70% EtOH/IMS 擦拭受影响区域。

Slow movement near and within the hood helps maintain constant circular airflow.
引擎盖附近和内部的缓慢移动有助于保持恒定的循环气流。

To help prevent contamination, as much as possible avoid speaking, sneezing and coughing while working in the hood.
为防止污染，在引擎罩内工作时应尽量避免说话、打喷嚏和咳嗽。

Discard pipette tips, waste reagents and waste medium into appropriately labelled beakers inside the hood. Discard used plasticware into the designated bins beside the hood.
将移液器吸头、废试剂和废培养基丢弃到罩内贴有相应标签的烧杯中。将用过的塑料器皿丢弃到罩旁的指定垃圾桶中。

When you have finished clear the hood and wipe the area with 70% EtOH/IMS. Dispose of waste appropriately. The hood should be left clean and ready for the next person to use.
完成后，清理通风橱，用 70% EtOH/IMS 擦拭该区域。妥善处理废弃物。通风橱应保持清洁，以备下一个人使用。

Making medium for cell culture
制作细胞培养基

Medium components:
中型组件：

- Dulbecco’s Modified Eagle’s medium (DMEM)
- 杜氏改良老鹰培养基（DMEM）

- Foetal calf serum (FCS)
- 胎牛血清（FCS）

-   Antibiotic-Antimycotic solution (100X stock: penicillin, streptomycin, amphotericin-B)
- 抗生素-抗霉菌药液（100X 储存液：青霉素、链霉素、两性霉素-B）

Calculate the volumes needed to make 50mls growth medium:
计算制作 50 毫升生长培养基所需的体积：

Demonstration
演示

- Setting up the tissue culture hood.
- 设置组织培养罩。

- Preparation of medium.
- 制备培养基。

- Plating cells from cryogenic stocks. Cryopreservation of cells allows stocks of cells to be stored for long (indefinite) periods of time. Cells are stored in liquid nitrogen.
- 从低温储存中培养细胞。细胞低温保存技术可使细胞储存长期（无限期）保存。细胞储存在液氮中。

Thaw cells from cryogenic stocks
解冻低温储存的细胞

Using aseptic technique make up 50mls medium using the volumes calculated. Make sure your medium tube is clearly labelled with your initials, date and contents as you will need this medium for Protocols 3 and 4.
使用无菌技术，按照计算的体积配制 50 毫升培养基。确保您的培养基试管上清楚地标有您的姓名首字母、日期和内容物，因为您需要用这种培养基来完成协议 3 和协议 4。

Prepare a 75cm² flask (T75) - take 10ml of the medium and put it into the flask.
准备一个 75 厘米 ² 烧瓶（T75）--取 10 毫升培养基放入烧瓶中。

Carefully remove a vial from liquid nitrogen using the correct PPE as instructed.
按照说明使用正确的个人防护设备，小心地从液氮中取出小瓶。

Thaw vial in a 37^oC water bath with gentle mixing until only the last small piece of ice is visible (hold the vial by the lid using the tips of your fingers only).
在 37 ^o C 水浴中轻轻搅拌解冻小瓶，直到只看到最后一小块冰为止（用手指尖夹住瓶盖）。

Take the vial to the hood.
把小瓶拿到通风橱中。

Using the pipettor with a 5ml pipette, take 1ml of medium from your flask, open the cryovial and gently suck up the cells into the liquid in the pipette. Avoid bubbles!
使用带有 5 毫升移液管的移液器，从烧瓶中取 1 毫升培养基，打开低温瓶，将细胞轻轻吸入移液管中的液体。避免产生气泡！

Decant all the liquid from the pipette into the flask and gently mix the cells and medium using the pipette to aspirate up and down gently to avoid creating bubbles.
将移液管中的所有液体倒入烧瓶中，用移液管轻轻地上下吸入，避免产生气泡，然后轻轻地混合细胞和培养基。

Label the flask with your name, the date, the name of the cells (copy what is written on the side of the cryovial).
在烧瓶上贴上标签，写上姓名、日期和细胞名称（复制低温瓶侧面的内容）。

Check the cells under the microscope so confirm there are cells present (cells will be floating initially but should attach to the substrate and spead).
在显微镜下检查细胞，确认是否有细胞存在（细胞最初会漂浮在水面上，但应附着在基质上并移动）。

Place the flask in the incubator making sure the flask is sitting flat on the shelf.
将烧瓶放入培养箱中，确保烧瓶平放在架子上。

Clean the hood – remove all items, disinfect the work area by wiping with 70% ethanol (EtOH) or industrial methylated spirit (IMS).
清洁引擎盖 - 取出所有物品，用 70% 乙醇 (EtOH) 或工业甲醇 (IMS) 擦拭工作区进行消毒。

Store any solutions you will need for subsequent protocols. Cell culture medium is generally stored at 4^oC (in a fridge or cold-room).
储存后续方案所需的任何溶液。细胞培养基通常储存在 4 ^o C（冰箱或冷藏室）中。

Protocol 3: Subculture (passaging) of adherent cells and cell counting
规程 3：粘附细胞的亚培养（传代）和细胞计数

Routine subculture of cells is required for maintenance of the culture, expanding the cell population, setting up cultures for experimental purposes and amplification of the stock for cryopreservation. For subculturing and counting adherent cells the cells must be detached from the substrate and dissociated into a single cell suspension. A variety of agents are used to detach cells from their substrate but a commonly used mixture is trypsin/EDTA. Trypsin is a protease and EDTA is a chelator that sequesters metal ions such as calcium and magnesium which cells require for adhesion to the substrate and to each other. In some cases, phenol red is added to the trypsin/EDTA solution as a pH indicator.
细胞的常规亚培养需要用于维持培养物、扩大细胞群、建立用于实验的培养物以及扩增用于低温保存的细胞储量。要对粘附细胞进行亚培养和计数，必须将细胞从基质上剥离并解离成单细胞悬浮液。有多种药剂可用于将细胞从基质上分离，但常用的混合物是胰蛋白酶/EDTA。胰蛋白酶是一种蛋白酶，而乙二胺四乙酸是一种螯合剂，可以螯合钙和镁等金属离子。在某些情况下，酚红被添加到胰蛋白酶/EDTA 溶液中作为 pH 指示剂。

A viable cell count is crucial for a variety of purposes eg. management of cell cultures and standardisation of cell concentrations at subculture, calculation of cell yields, titration of cell populations in diagnostics, and in industrial bioprocesses. Cells can be counted using automated cell counters or manually using a haemocytometer. Although cell counters are fast, reliable and less prone to operator error, counters and consumables are expensive and manual counting with a haemocytometer is still commonly used due to the low cost and versatility.
存活细胞计数对多种用途都至关重要，例如细胞培养管理、亚培养细胞浓度标准化、细胞产量计算、诊断中细胞群滴定以及工业生物处理。细胞计数可使用自动细胞计数器或手动血细胞计数器。虽然细胞计数器快速、可靠且不易出现操作错误，但计数器和耗材价格昂贵，使用血细胞计数器进行手动计数因其成本低、用途广而仍被广泛使用。

Trypsinisation of cells
细胞胰蛋白酶化

Under the microscope check the cells, in your lab book make a note of the cell morphology - and assess the degree of confluency (ie. the % surface area covered by cells).
在显微镜下检查细胞，在实验记录本上记录细胞形态--并评估汇合程度（即细胞覆盖的表面积百分比）。

Take a photo of your cells.
给细胞拍照。

Cells are usually sub-cultured at around 80% confluency.
细胞通常在 80% 左右的融合度时进行亚培养。

In the hood:
在引擎盖里

Aspirate medium from the flask taking care not to scratch the cell monolayer with the pipette.
吸出烧瓶中的培养基，注意不要用移液管刮伤细胞单层。

Wash cells with 10mls sterile Dulbecco’s modified phosphate-buffered saline (PBS) to remove residual serum which will neutralise protease activity.
用 10 毫升无菌杜尔贝科改良磷酸盐缓冲盐水（PBS）清洗细胞，以去除会中和蛋白酶活性的残留血清。

Aspirate all the PBS.
吸出所有 PBS。

Add 2ml trypsin-EDTA.
加入 2 毫升胰蛋白酶-EDTA。

Gently swirl to ensure that the cells are covered by trypsin-EDTA.
轻轻旋转，确保细胞被胰蛋白酶-EDTA 覆盖。

Incubate at 37^oC (2 mins initially but times will vary depending on cell type and degree of confluency, check every 2 mins). Note: Prolonged exposure of cells to trypsin-EDTA can damage the cells.
在 37 ^o C 温度下孵育（初始孵育时间为 2 分钟，孵育时间因细胞类型和汇合程度而异，每 2 分钟检查一次）。注意：细胞长时间暴露于胰蛋白酶-EDTA 会损伤细胞。

Monitor detachment over time by checking under the microscope.
通过在显微镜下检查来监测脱落情况。

Once cells have detached and formed a single cell suspension (clumps make it impossible to get an accurate cell count), add 5ml of growth medium (from Protocol 2) to neutralise the protease. Mix well by gently pipetting up and down. Decant the cell suspension to a 15ml tube.
细胞脱落并形成单个细胞悬浮液后（细胞团块会导致无法获得准确的细胞数），加入 5 毫升生长培养基（步骤 2 中的培养基）以中和蛋白酶。上下轻轻移液，充分混合。将细胞悬浮液倒入 15 毫升的试管中。

Trypan blue exclusion test to assess cell viability: The dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells have intact cell membranes that exclude certain dyes, such as Trypan blue, whereas dead cells do not. In this test, a cell suspension is simply mixed with Trypan blue and then microscopically examined to determine whether cells take up the dye (dead cells) or not (viable cells).
胰蓝排除试验评估细胞活力：染料排阻试验用于确定细胞悬液中有活力细胞的数量。其原理是活细胞的细胞膜完整，能排除某些染料，如 Trypan 蓝，而死细胞则不能。在该试验中，只需将细胞悬液与 Trypan 蓝混合，然后用显微镜检查，即可确定细胞是否吸收染料（死细胞）（活细胞）。

Haemocytometer: The haemocytometer was originally invented for blood cell counting. The gridded area of the haemocytometer consists of nine 1 x 1 mm (1 mm²) squares. These nine squares are further divided into smaller squares. The large corner squares (subdivided into 16 squares) are used for cell counting in our experiment. The raised edges of the haemocytometer hold the coverslip 0.1 mm off the marked grid, giving each large square a defined volume. The volume of each corner square is 100nl (10^-4 ml).
血细胞计数器血细胞计数器最初是为血细胞计数而发明的。血细胞计数器的网格区域由九个 1 x 1 毫米（1 毫米 ² ）的方格组成。这九个方格又被进一步划分为更小的方格。在我们的实验中，大的角方格（细分为 16 个方格）用于细胞计数。血细胞计数器凸起的边缘将盖玻片固定在离标记网格 0.1 毫米的地方，使每个大方格都有确定的体积。每个角方格的体积为 100n（10 ^-4 m）。

Aliquot 20µl of the cell suspension to a 1.5ml microcentrifuge tube. Note: Mix the cell suspension well before taking the aliquot to ensure cells are evenly distributed
取 20µl 细胞悬浮液到 1.5 毫升微离心管中。注意：取等分试样前充分混合细胞悬液，以确保细胞分布均匀。

Add 20µl Trypan blue dye (Cells:Trypan blue, ratio 1:1).
加入 20 微升胰蓝染料（细胞：胰蓝，比例 1:1）。

Mix gently.
轻轻搅拌。

Load cells into the haemocytometer,10µl/chamber.
将细胞装入血细胞计数器，10 微升/室。

Count cells and record the number of viable cells and non-viable cells. Usually the 4 corner chambers are counted. Viable cells are bright (as they exclude the dye), dead cells are dark (as they take up the dye). Make a note of your cell counts in your lab book.
计数细胞并记录存活细胞和未存活细胞的数量。通常对 4 个角室进行计数。存活的细胞是亮的（因为它们排除了染料），死亡的细胞是暗的（因为它们吸收了染料）。在实验本上记下细胞数。

Ideally ~100 cells/large square should be counted to increase the accuracy of the cell count.
理想情况下，每个大方格应计数 ~100 个细胞，以提高细胞计数的准确性。

Check your cell counts with a demonstrator to see how close the counts are to their count.
与演示者核对细胞数，看看与他们的细胞数有多接近。

Calculate the concentration of viable and non-viable cells and the % of viable cells using the following equations where:
用以下公式计算存活细胞和未存活细胞的浓度以及存活细胞的百分比：

A = Mean number of viable cells counted
A = 计数的存活细胞平均数

Total number of viable cells counted/number of squares
计数的存活细胞总数/方格数

B = Mean number of non-viable cells counted
B = 无活力细胞的平均计数

Total number of non- viable cells counted/number of squares
计数的非存活细胞总数/方格数

C = Dilution factor
C = 稀释因子

D = Correction factor
D = 校正系数

The area of the large square (16 smaller squares) is 1mm² and the depth of the chamber is
大正方形（16 个小正方形）的面积为 1 毫米 ² ，室的深度为

0.1mm
0.1 毫米

The correction factor of 10⁴ converts 0.1mm³ to 1ml
修正系数 10 ⁴ 将 0.1 毫米 ³ 转换为 1 毫升

Concentration of viable cells (cells/ml) = A x C x D
存活细胞浓度（细胞/毫升）= A x C x D

Concentration of non-viable cells (cells/ml) = B x C x D
无活力细胞浓度（细胞/毫升）= B x C x D

Total number of viable cells = concentration of viable cells x volume
存活细胞总数 = 存活细胞浓度 x 体积

Total number of cells = number of viable cells + non number of non-viable cells
细胞总数 = 有活力细胞数 + 无活力细胞数

% viability = No. of viable cells x 100
存活率 = 存活细胞数 x 100
Total number of cells
细胞总数

Possible sources of inaccuracy in the count:
计数不准确的可能原因：

Bubbles and debris in the chamber.
舱内有气泡和碎片。

Overfilling the chamber such the sample runs into the channels or the other chamber
样品室填充过满，导致样品流入通道或另一个样品室

Incomplete filling of the chamber.
气室填充不完全。

Cells not evenly distributed throughout the chamber and/or not in a single cell suspension.
细胞未均匀分布在整个室中，和/或不是单细胞悬浮液。

Too few cells to count. If necessary, this can be overcome by centrifuging the cells, resuspending in a smaller volume and recounting.
细胞太少，无法计数。如有必要，可将细胞离心，以较小体积重新悬浮，然后重新计数。

Too many cells to count. This can be overcome by using a higher dilution factor in Trypan Blue e.g. 1 volume cells:10 volumes TB.
细胞太多，无法计数。可通过提高胰蓝的稀释倍数来解决这一问题，例如 1 体积细胞：10 体积 TB。

Passaging cells
传代细胞

Calculate the number of cells needed to plate all the wells of a 6-well plate at 0. 5x10⁶ cells/well.
计算在 6 孔板的所有孔中，以 0.5x10 ⁶ 个细胞/孔的条件进行培养所需的细胞数。

Note: In a 6-well plate each well contains 2ml of medium so you want 12mls at 0.25x10⁶ cells/ml.
注：在 6 孔板中，每孔含有 2 毫升培养基，因此需要 12 毫升，细胞数为 0.25x10 ⁶ 个/毫升。

Calculate the volume of the cell suspension you will need and aliquot the volume into a 15ml tube (ensure to mix the cell suspension by repeated inversion before taking the aliquot as cells will sink to the bottom of the tube over time e.g. while you are counting cell numbers).
计算所需细胞悬浮液的体积，然后将其等分到 15 毫升的试管中（确保在取等分试管前反复倒置细胞悬浮液以混合细胞，因为细胞会随着时间沉入试管底部，例如在计算细胞数时）。

Make the volume up to 12ml total volume with growth medium.
用生长培养基将总体积增大到 12 毫升。

Plate cells into a 6 well plate, 2ml/well.
将细胞培养到 6 孔板中，每孔 2 毫升。

Label the plate with the cell type, date and your name.
在平板上标注电池类型、日期和您的姓名。

Check under the microscope. By eye, do the wells contain similar numbers of cells?
在显微镜下检查。目测各孔中的细胞数量是否相似？

Place the plate in the incubator, make sure the plate is sitting flat on the shelf. Grow at 37^oC in 5% CO₂.
将平板放入培养箱，确保平板平放在架子上。在 37 ^o C 温度、5% CO ₂ 条件下生长。

Protocol 4: Treatment of cells with Transforming growth factor (TGF)b and harvesting for gene and protein expression analyses
方案 4：用转化生长因子（TGF）b 处理细胞并收获细胞进行基因和蛋白质表达分析

Cell cultures are frequently used to model disease. For example, a commonly used model of fibrotic disease is to treat cells with known fibrogenic factors such as Transforming growth factor (TGF)b or Platelet-derived growth factor (PDGF) and compare gene and protein expression in treated versus vehicle-treated (control) cells. Note: The vehicle is whatever the solution is that is used to reconstitute/dilute the growth factor. Control cells are treated with an equivalent volume of vehicle to eliminate the possibility that any observed changes are due to the vehicle rather than to the growth factor.
细胞培养常用于疾病模型。例如，常用的纤维化疾病模型是用已知的纤维化因子（如转化生长因子（TGF）b 或血小板衍生生长因子（PDGF））处理细胞，然后比较处理细胞与载体处理（对照）细胞的基因和蛋白质表达。注：载体是指用于重组/稀释生长因子的溶液。对照组细胞用等体积的载体处理，以消除观察到的任何变化是由载体而非生长因子引起的可能性。

Using the cells in a 6-well plate from Protocol 3, you will treat half the cells (3 wells) with TGFb and half with the same medium containing an equivalent volume of vehicle (vehicle treated/control). For treatment with specific growth factors, the FCS in the medium is either removed or severely reduced e.g. to 0.5-1%, so it is possible to study the effect of specific factors without the confounding influence of undefined serum factors.
使用方案 3 中 6 孔板中的细胞，用 TGFb 处理一半细胞（3 孔），用含有等量载体的相同培养基处理一半细胞（载体处理/对照）。在使用特定生长因子处理时，培养基中的 FCS 要么去除，要么严重降低，例如降至 0.5-1%，这样就可以研究特定因子的影响，而不会受到未定义的血清因子的干扰。

Prepare 50ml DMEM containing 0.5% FCS and 1% antibiotic/antimycotic as for Protocol 2:
按步骤 2 的方法配制 50 毫升含 0.5% FCS 和 1% 抗生素/抗霉菌素的 DMEM：

Prepare medium with and without TGFb 2ng/ml:
制备含 TGFb 2ng/ml 和不含 TGFb 2ng/ml 的培养基：

Aliquot 7mls of medium into two 15 ml tubes, label the tubes “TGFb” and “Vehicle”.
取 7 毫升培养基放入两个 15 毫升的试管中，分别标上 "TGFb "和 "载体"。

Note: When making up solutions for treating cells it is advisable to make a little more than is needed to ensure you have enough to treat all the wells with the full volume.
注：在配制处理细胞的溶液时，最好比需要的量多一点，以确保有足够的溶液处理所有孔。

You have a stock of TGFb 2ng/ml in 4mM HCl, 1mg/ml bovine serum albumin (BSA) and a stock of 4mM HCl, 1mg/ml BSA (vehicle).
您有 2ng/m 的 TGFb（4mM HCl、1mg/ml 牛血清白蛋白 (BSA)）和 4mM HCl、1mg/ml BSA（载体）。

Calculate the volume of TGFb you will need to add to 7mls to give a final concentration of 2ng/ml.
计算在 7 毫升中添加 TGFb 的体积，以获得 2ng/ml 的最终浓度。

Add TGFb to the 7mls medium, close the tube tightly and mix by inverting gently.
在 7 毫升培养基中加入 TGFb，盖紧试管并轻轻倒置混合。

Add an equivalent volume of vehicle to the second tube and mix.
在第二支试管中加入等体积的载体并混合。

Treating cells
治疗细胞

Collect your 6-well plate from the incubator, check the cells under the microscope – make a note of the morphology and the degree of confluency and take a photo.
从培养箱中取出 6 孔板，在显微镜下检查细胞--记下形态和融合度并拍照。

Note: When washing and treating the cells make sure the plate is kept covered as much as possible to minimise the chance of the cells drying out.
注意：在清洗和处理细胞时，确保尽量盖住平板，以减少细胞干涸的机会。

Aspirate the medium from the wells and add DMEM + 0.5% FSC+1% antibiotic/antimycotic 2mls/well, gently rock the plate to wash the cells.
吸干孔中的培养基，加入 DMEM + 0.5% FSC+1% 抗生素/抗霉菌剂 2 毫升/孔，轻轻摇动平板清洗细胞。

Aspirate the medium.
吸出培养基。

Add the appropriate medium to the wells, 2ml/well.
在孔中加入适当的培养基，每孔 2 毫升。

Label the plate to show, 3 wells “Vehicle” or “-“ i.e. no TGFb and 3 wells “TGFb” or “+” so you know what the cells in each well have been treated with.
给平板贴上标签，显示 3 个孔为 "载体 "或"-"，即不含 TGFb，3 个孔为 "TGFb "或 "+"，这样就可以知道每个孔中的细胞用什么处理过。

Check the cells under the microscope (to ensure you still have healthy cells attached to the plate!).
在显微镜下检查细胞（确保仍有健康的细胞附着在平板上！）。

Replace the plate in the incubator, again ensuring the plate is sitting flat on the shelf.
将平板放回培养箱，再次确保平板平放在架子上。

Harvesting cells for RNA and protein expression analyses
收获细胞进行 RNA 和蛋白质表达分析

After the appropriate incubation time (generally 24-72 hours), vehicle-treated and TGFb-treated cells are collected for analysis of RNA and protein expression.
经过适当的培养时间（一般为 24-72 小时）后，收集经车辆处理和经 TGFb 处理的细胞，以分析 RNA 和蛋白质的表达。

Remove the plate from the incubator and check the cells under the microscope, make a note of the morphology and the degree of confluency of the vehicle-treated and TGFb-treated cells. Are there any differences between the two groups? You may want to take a photo.
将培养皿从培养箱中取出，在显微镜下检查细胞，记录载体处理过的细胞和 TGFb 处理过的细胞的形态和融合度。两组细胞之间是否存在差异？您可能需要拍摄照片。

Place the 6-well plate on ice and remove the medium.
将 6 孔板放在冰上，取出培养基。

Wash the cells twice with ice-cold PBS (2ml/well).
用冰冷的 PBS（2 毫升/孔）清洗细胞两次。

Leave the cells covered with ice-cold PBS to avoid cells drying out as wells will be processed at slightly different times.
用冰冷的 PBS 盖住细胞，避免细胞干燥，因为各孔的处理时间略有不同。

RNA sample preparation
RNA 样品制备

Efficient disruption and homogenisation of the starting material is essential for RNA purification.
有效地破坏和均质化起始材料对 RNA 纯化至关重要。

Complete disruption of cell walls and plasma membranes of cells and organelles is essential to release all the RNA contained in the sample. Incomplete disruption results in significantly reduced RNA yields. Homogenization reduces the viscosity of the lysates by shearing high molecular-weight genomic DNA and other high molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in inefficient binding of RNA to the spin column membrane and therefore significantly reduces RNA yield.
完全破坏细胞壁以及细胞和细胞器的质膜对于释放样本中的所有 RNA 至关重要。不完全破坏会导致 RNA 产量大大降低。均质化可通过剪切高分子量基因组 DNA 和其他高分子量细胞成分来降低裂解液的粘度，从而形成均匀的裂解液。不完全均质化会导致 RNA 与旋转柱膜的结合效率低下，从而大大降低 RNA 产量。

Note: When extracting and working with RNA, RNAse-free solutions and equipment must be used to minimize degradation of RNA by environmental RNases. One of the major sources of RNase is your hands, make sure you wear gloves!
注意：提取和处理 RNA 时，必须使用不含 RNAse 的溶液和设备，以尽量减少环境中 RNase 对 RNA 的降解。手是 RNase 的主要来源之一，请务必戴上手套！

Total RNA will be extracted using the RNeasy Mini kit from Qiagen (see Protocol 5). Samples are first lysed and homogenised in highly denaturing guanidine thiocyanate-containing buffer which inactivates any RNAses.
总 RNA 将使用 Qiagen 公司的 RNeasy Mini 试剂盒提取（见步骤 5）。样品首先在含硫氰酸胍的高变性缓冲液中裂解和均质，使任何 RNAses 失活。

Tilt the plate and remove all the PBS from one “Vehicle” well and one “TGFb” well.
倾斜平板，从一个 "车辆 "孔和一个 "TGFb "孔中移除所有 PBS。

Add 600ml Buffer RLT (lysis buffer) and scrape the cells off the plate using a cell scraper.
加入 600 毫升缓冲液 RLT（裂解缓冲液），用细胞刮板将细胞从平板上刮下。

Transfer the lysate to a clean RNAse-free 1.5ml microcentrifuge tube and vortex.
将裂解液转移到干净的不含 RNAse 的 1.5 毫升微离心管中并涡旋。

Homogenise the lysate using a 20gauge (20G) needle attached to a plastic syringe. The lysate is gently passed through the needle 5-10X until a homogeneous lysate is obtained. Note: Take care not to create bubbles.
使用连接塑料注射器的 20 号（20G）针头均质裂解液。将裂解液轻轻穿过针头 5-10 倍，直至获得均匀的裂解液。注意：小心不要产生气泡。

Check by eye that the two samples have a similar volume.
用眼睛检查两个样品的体积是否相似。

Store the lysate at -20^oC or -70^oC for subsequent use.
将裂解液储存在 -20 ^o C 或 -70 ^o C 以备后用。

Protein sample preparation
蛋白质样品制备

Tilt the plates at an angle and remove all the PBS from one “Vehicle” well and one “TGFb” well.
将平板倾斜，从一个 "车辆 "孔和一个 "TGFb "孔中取出所有 PBS。

Add 175µl lysis buffer (RIPA Buffer: 50 mM Tris-HCL, pH8.0 with 150 mM sodium chloride, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1X protease and phosphatase inhibitors) to the cells and harvest using a cell scraper.
加入 175µl 裂解缓冲液（RIPA 缓冲液：50 mM Tris-HCL，pH8.0，含 150 mM 氯化钠、1% Igepal CA-630、0.5% 脱氧胆酸钠、0.1% SDS、1 倍蛋白酶和磷酸酶抑制剂），然后用细胞刮刀刮取细胞。

Transfer the cell lysate to a 1.5 ml microcentrifuge tube and spin at maximum speed in a microfuge for 10 minutes at room temperature to pellet any insoluble material.
将细胞裂解液转移到 1.5 毫升的微离心管中，在室温下以最大速度在微量离心管中旋转 10 分钟，将所有不溶性物质沉淀下来。

Transfer the supernatant to a clean tube, taking care not to disrupt the pellet.
将上清液转移到干净的试管中，注意不要破坏颗粒。

Check by eye that the two samples have a similar volume.
用眼睛检查两个样品的体积是否相似。

Store the supernatant at -20^oC for subsequent use. Discard the pellet.
将上清液储存在 -20 ^o C 以备后用。丢弃颗粒。

Samples can be stored at -70^oC for longer term storage.
样品可在 -70 ^o C 温度下长期保存。

Protocol 5: Extraction of RNA from LX2 cells using the RNeasy Mini Kit
规程 5：使用 RNeasy Mini Kit 提取 LX2 细胞中的 RNA

(QIAGEN Catalogue number: 74104)
(QIAGEN 目录号：74104）。

The RNeasy procedure represents a well-established technology for RNA purification. This technology combines the selective binding properties of a silica-based membrane with the speed of microspin technology. A specialized high-salt buffer system allows up to 100 µg RNA to bind to the RNeasy silica membrane. Biological samples are first lysed and homogenized in the presence of a highly denaturing guanidine-thiocyanate–containing buffer, which immediately inactivates RNAses to ensure purification of intact RNA. Ethanol is added to provide appropriate binding conditions, and the sample is then applied to a RNeasy Mini spin column, where the total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA is then eluted in 30–100 µl water. With the RNeasy procedure, all RNA molecules longer than 200 nucleotides are purified. The procedure provides an enrichment for mRNA since most RNAs <200 nucleotides (such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise 15–20% of total RNA) are selectively excluded.
RNeasy 程序是一种成熟的 RNA 纯化技术。该技术结合了硅胶膜的选择性结合特性和微旋技术的速度。专用的高盐缓冲液系统可使高达 100 µg 的 RNA 与 RNeasy 硅胶膜结合。生物样本首先在含有高变性胍-硫氰酸盐的缓冲液中裂解和匀浆，该缓冲液可立即使 RNAses 失活，以确保纯化完整的 RNA。加入乙醇以提供适当的结合条件，然后将样品加入 RNeasy Mini 纺丝柱，总 RNA 与膜结合，杂质被有效洗去。然后用 30-100 µl 的水洗脱高质量的 RNA。通过 RNeasy 程序，可纯化所有长度超过 200 个核苷酸的 RNA 分子。由于大多数核苷酸小于 200 个的 RNA（如 5.8S rRNA、5S rRNA 和 tRNA，它们共占总 RNA 的 15-20%）都被选择性地排除在外，因此该程序可富集 mRNA。

Protocols for purification of RNA using RNeasy Kits are available at:
使用 RNeasy 试剂盒纯化 RNA 的方法可在以下网站获取：

https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-mini-kit/?clear=true#productdetails

Flowchart for the protocol from Qiagen handbook:
Qiagen 手册中的方案流程图：

Protocol
规程

The following protocol is for cells from 1 well of a 6-well plate of confluent adherent cells. Each member of the pair can do one sample (1 well).
以下操作步骤适用于 6 孔板中 1 孔的汇合粘附细胞。每个配对成员可做一个样品（1 孔）。

Make sure you know which sample is Vehicle and which is TGFb - MAKE SURE TUBES ARE LABELLED!
确保您知道哪个样本是 Vehicle，哪个是 TGFb - 确保试管贴有标签！

Thaw the lysates from Protocol 4 at room temperature until completely thawed and salts are dissolved. Avoid prolonged incubation, which may compromise RNA integrity. If any insoluble material is visible, centrifuge for 5 min at maximum in a microfuge and transfer supernatant to a new RNAse-free tube.
在室温下解冻步骤 4 中的裂解液，直至完全解冻且盐分溶解。避免长时间孵育，以免影响 RNA 的完整性。如果发现任何不溶性物质，在微量离心机中离心 5 分钟，并将上清液转移到一个新的不含 RNAse 的试管中。

Measure the volume of the lysate in the tube, add an equal volume of 70% EtOH and mix well by pipetting.
测量试管中裂解液的体积，加入等体积的 70% EtOH，用移液器混合均匀。

Place a RNeasy spin column (pink column) in a 2 ml collection tube, one column for each sample. Make sure the columns are labelled so you know which sample is in which column. Transfer half the sample (maximum volume 700ml) to the column taking care not to touch the membrane at the base of the column. Place the column/collection tube in the centrifuge close the lid gently and centrifuge for 15 seconds at 9000 x g. Discard the flow-through ie. the solution what is in the collection tube. Repeat, this step so the whole sample passes through the column. Discard the flow-through.
将 RNeasy 自旋柱（粉红色柱）放入 2 毫升的收集管中，每个样本一个柱。确保柱子上贴有标签，以便知道哪个样品在哪个柱子里。将一半的样品（最大容量 700 毫升）转移到柱中，注意不要碰到柱底部的膜。将色谱柱/收集管放入离心机中，轻轻盖上盖子，在 9000 x g 转速下离心 15 秒。重复此步骤，使整个样品通过色谱柱。丢弃流出液。

Add 700µl Buffer RW1 to the RNeasy spin column. Close the centrifuge lid gently and centrifuge for 15 seconds at maximum speed to wash the spin column membrane. Discard the flow-through.
向 RNeasy 自旋柱中加入 700µl 缓冲液 RW1。轻轻盖上离心机盖，以最高转速离心 15 秒钟，以清洗旋柱膜。弃去流出液。

Add 500µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 seconds at maximum speed to wash the spin column membrane. Discard the flow-through.
向 RNeasy 自旋柱中加入 500µl 缓冲液 RPE。轻轻盖上盖子，以最大速度离心 15 秒钟，以清洗旋柱膜。弃去流出液。

Add 500µl Buffer RPE to the RNeasy spin column. Close the lid gently and centrifuge for 1 minute at maximum speed. Discard the flow-through.
在 RNeasy 旋转柱中加入 500µl 缓冲液 RPE。轻轻盖上盖子，以最高转速离心 1 分钟。弃去流出液。

Spin for 3 minutes to completely dry the membrane. This last longer centrifugation step dries the spin column membrane, ensuring that no EtOH is carried over during the elution step. After centrifugation, carefully remove the RNeasy spin column from the collection tube making sure that the column does not contact the flow-through to avoid any carry-over of EtOH.
离心 3 分钟，使膜完全干燥。这最后一个较长的离心步骤可使自旋柱膜干燥，确保在洗脱步骤中没有 EtOH 残留。离心后，小心地将 RNeasy 自旋柱从收集管中取出，确保自旋柱不与流出液接触，以避免 EtOH 残留。

Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30µl RNase-free water directly onto the centre of the spin column membrane. Note: Do NOT touch the membrane with the pipette tip. Close the lid gently, leave to stand for 1 minute at room temperature then centrifuge for 1 minute at full speed to elute the RNA.
将 RNeasy 自旋柱放入新的 1.5 毫升收集管中。将 30µl 不含 RNase 的水直接加到自旋柱膜中心。注意：不要用移液器吸头接触膜。轻轻盖上盖子，在室温下静置 1 分钟，然后全速离心 1 分钟以洗脱 RNA。

Use a small aliquot (1-2ml) of the RNA to measure the concentration (see below). The remainder should be stored either on ice if it’s to be used immediately or for longer storage store at -20°C or -70°C.
取一小部分（1-2 毫升）RNA 来测量浓度（见下文）。如果要立即使用，剩余部分应冰冻保存；如果要长期保存，则应保存在 -20°C 或 -70°C。

RNA concentration and purity are assessed using a microvolume spectrophotometer called a Nanodrop (Thermo Scientific) which shows the sample absorbance profile. Make sure you record these values.
RNA 的浓度和纯度可通过一种名为 Nanodrop（Thermo Scientific 公司）的微量分光光度计来评估，该仪器可显示样品的吸光度曲线。请务必记录这些值。

Measuring the concentration and purity of RNA
测量 RNA 的浓度和纯度

The concentration of RNA in a pure sample can be measured using a spectrophotometer. The figure below shows maximum absorption at 260 nm. Therefore, the concentration of RNA can be determined by measuring the absorbance of the diluent at 260nm (A260). To ensure the sample is pure, the absorption can be measured at two separate wavelengths and the ratio compared to the ratio of absorption expected from a pure RNA sample. Purity of RNA is given by the A260/A280 and A260/A230 (A=Absorbance) ratios.
纯样品中的 RNA 浓度可用分光光度计测量。下图显示了 260 纳米波长处的最大吸收率。因此，可通过测量稀释液在 260 纳米波长处的吸光度（A260）来确定 RNA 的浓度。为确保样品的纯度，可分别在两个波长处测量吸光度，并将其比值与纯 RNA 样品的预期吸光度比值进行比较。RNA 的纯度由 A260/A280 和 A260/A230（A=吸光度）比值决定。

A260/A280 of 1.8 - 2.0 and/or A260/A230 of 2.0 - 2.2 for RNA is considered pure.
A260/A280 为 1.8 - 2.0 和/或 A260/A230 为 2.0 - 2.2 的 RNA 被认为是纯的。

A260/A280 lower than expected often indicates contamination with protein.
A260/A280 低于预期值通常表明受到蛋白质污染。

A260/A230 lower than expected often indicates contamination with salt (such as guanidine used in the kit).
A260/A230 低于预期通常表明受到盐（如试剂盒中使用的胍）污染。

We will be using a Nanodrop which is a spectrophotometer that gives the absorbance profile of RNA, DNA and proteins, based on the absorbance at different wavelengths. Use of the instrument will be demonstrated for you first and then you can analyse your sample.
我们将使用 Nanodrop 分光光度计，它可以根据不同波长的吸光度显示 RNA、DNA 和蛋白质的吸光度曲线。我们会先向您演示仪器的使用方法，然后您就可以分析自己的样品了。

Using a Nanodrop Spectrophotometer:
使用 Nanodrop 分光光度计：

1. From the home screen, select the Nucleic Acid tab and tap “RNA”.
1.从主屏幕选择 "核酸 "选项卡，然后点击 "RNA"。

2. To blank the machine, pipette 2µl of RNase-free water onto the lower pedestal and lower the arm.
2.要对机器进行空白处理，请在下基座上吸入 2 微克不含 RNase 的水，然后放下手臂。

3. Tap “Blank” and wait for the measurement to complete.
3.点击 "空白"，等待测量完成。

4. Lift the arm and clean both pedestals with a clean laboratory wipe.
4.抬起机械臂，用干净的实验室抹布清洁两个基座。

5. To measure samples, pipette 2µl of sample onto the pedestal and lower the arm, tap “Measure”.
5.测量样品时，在基座上移取 2 微米的样品，然后放下测量臂，点击 "测量"。

6. Clean the pedestal with a clean wipe and repeat step 5 for the next sample.
6.用干净的抹布擦拭基座，然后重复步骤 5 取下一个样品。

7. Record your result in your lab book using the table shown below:
7.用下表将结果记录在实验本上：

8. When you have finished measuring samples, tap “End Experiment”.
8.完成样品测量后，点击 "结束实验"。

9. Lift the arm and clean the pedestal with a clean wipe.
9.抬起机械臂，用干净的抹布清洁基座。

Make a note of your interpretation of what the concentrations and A260/A280 and A260/A230 ratios mean as they relate to your samples.
记下您对浓度、A260/A280 和 A260/A230 比率的理解，因为它们与您的样品有关。

If ratios are low, make a note in your lab-book of possible causes of reduced RNA purity?
如果比率较低，请在实验手册中注明导致 RNA 纯度降低的可能原因？

Protocol 6: Reverse transcription using QuantiTect Reverse Transcription Kit (QIAGEN Catalogue number: 205311)
步骤 6：使用 QuantiTect 反转录试剂盒（QIAGEN 目录号：205311）进行反转录

Background
背景介绍

The QuantiTect Reverse Transcription Kit is designed for use in two-step RT-PCR and provides high cDNA yields for sensitive quantification of even low-abundance transcripts.
QuantiTect 逆转录试剂盒设计用于两步 RT-PCR，可提供高 cDNA 产量，即使是低丰度转录本也能灵敏定量。

The procedure comprises 2 main steps (see Protocol flowchart below):
该程序包括 2 个主要步骤（见下面的协议流程图）：

Elimination of genomic DNA
消除基因组 DNA

Genomic DNA contamination in RNA samples must be eliminated to avoid detection of genomic DNA rather than cDNA.
必须消除 RNA 样品中的基因组 DNA 污染，以避免检测到基因组 DNA 而非 cDNA。

Reverse transcription
反转录

After removal of genomic DNA, the RNA sample is reverse transcribed using a Master Mix containing Quantiscript Reverse Transcriptase, Quantiscript RT Buffer and RT Primer Mix. Quantiscript Reverse Transcriptase has a high affinity for RNA and is optimized for efficient and sensitive cDNA synthesis from 10pg - 1μg of RNA. High RNA affinity, enables high cDNA yields, even from templates with high GC-content or complex secondary structure. RT Primer Mix ensures cDNA synthesis from all regions of RNA transcripts, even from 5' regions. This allows high yields of cDNA template for PCR analysis regardless of where the target region is located in the transcript.
去除基因组 DNA 后，使用含有 Quantiscript 逆转录酶、Quantiscript RT 缓冲液和 RT 引物混合液的主混合物对 RNA 样品进行逆转录。Quantiscript 逆转录酶对 RNA 有很高的亲和力，经过优化可从 10pg - 1μg RNA 中高效灵敏地合成 cDNA。高 RNA 亲和力使得即使是高 GC 含量或复杂二级结构的模板也能获得高产率的 cDNA。RT 引物混合液可确保从 RNA 转录本的所有区域，甚至从 5' 区域合成 cDNA。这样，无论目标区域位于转录本的哪个位置，都能获得高产率的 cDNA 模板，用于 PCR 分析。

Protocol
规程

QuantiTect Reverse Transcription Procedure
QuantiTect 逆转录程序

The following procedure is designed to convert 10pg - 1 µg of total RNA into cDNA:
以下程序旨在将 10pg - 1 µg 的总 RNA 转化为 cDNA：

Label a 1.5ml tube for each of your samples.
为每个样品的 1.5 毫升试管贴上标签。

Calculate the volume of sample you need for 1mg RNA.
计算 1 毫克 RNA 所需的样品量。

Mix and briefly centrifuge each component before use.
使用前将各成分混合并短暂离心。

Combine the following:
结合以下内容：

Incubate at 42°C for 2 minutes, then place on ice for at least 1 minute.
在 42°C 孵育 2 分钟，然后放在冰上至少 1 分钟。

Centrifuge briefly to remove any condensation from the lid of the tube.
短暂离心，除去试管盖上的冷凝水。

Prepare the following cDNA Synthesis mix, adding each component in the indicated order:
准备以下 cDNA 合成混合物，按指定顺序添加各组分：

.

Incubate as follows:
孵育方法如下：

 - 15 minutes at 42°C.
- 15 分钟，温度 42°C。

  - 3 minutes at 95°C, to inactivate Quantiscript Reverse Transcriptase.
- 95°C 3 分钟，灭活 Quantiscript 逆转录酶。

- Chill on ice.
- 冰镇

cDNA synthesis reaction can be used for PCR immediately or stored at -20°C for future use.
cDNA 合成反应物可立即用于 PCR，也可保存在 -20°C 以备将来使用。

If the cDNA is to be used for PCR immediately as is the case here, dilute 1:10 in DNAse-free water.
如果 cDNA 要立即用于 PCR（如本例），则用无 DNAse 水以 1:10 稀释。

Protocol 7: Polymerase Chain Reaction using AllTaq DNA polymerase (Qiagen Catalogue number: 203144)
规程 7：使用 AllTaq DNA 聚合酶（Qiagen 目录编号：203144）进行聚合酶链反应

Polymerase Chain Reaction (PCR) is a technique used to rapidly produce (amplify) multiple copies of a specific DNA sequence. PCR uses DNA polymerase, primers, and dNTPs to amplify the sequence on a DNA Template. By changing the temperature of the reaction mixture, the activity of the polymerase is controlled.
聚合酶链式反应（PCR）是一种用于快速产生（扩增）多个特定 DNA 序列拷贝的技术。PCR 使用 DNA 聚合酶、引物和 dNTP 来扩增 DNA 模板上的序列。通过改变反应混合物的温度，可以控制聚合酶的活性。

After the purification of total RNA and reverse transcription into cDNA, a PCR reaction will be performed to investigate the expression of Collagen 1 a major component of the extracellular matrix (ECM) in LX2 cells and determine whether expression is regulated by TGFb in this cell type. PCR requires a pair of primers (forward (F) and reverse (R)).
纯化总 RNA 并反转录为 cDNA 后，将进行 PCR 反应，以研究 LX2 细胞中细胞外基质 (ECM) 的主要成分胶原 1 的表达情况，并确定该细胞类型中胶原 1 的表达是否受 TGFb 的调控。PCR 需要一对引物（正向引物 (F) 和反向引物 (R)）。

The sequence of the primers for amplification of the Collagen 1 (COL1A2) transcript are:
用于扩增胶原蛋白 1 (COL1A2) 转录本的引物序列为

As a positive control a “housekeeping” or constitutively-expressed gene is also used. A frequently used gene is TBP (TATA box binding protein) a general transcription factor that is a key component of the eukaryotic transcription machinery.
还可使用 "看家 "基因或组成型表达基因作为阳性对照。一个常用的基因是 TBP（TATA 盒结合蛋白），它是一种通用转录因子，是真核生物转录机制的关键组成部分。

The sequence of the primers for amplification of the TBP transcript are:
用于扩增 TBP 转录本的引物序列为

Notes
说明

AllTaq DNA Polymerase requires a heat-activation step of 2 minutes at 95°C or 3 min at
AllTaq DNA 聚合酶需要 95°C 下 2 分钟或 3 分钟的热激活步骤。

93°C for long amplicons.
93°C 用于长扩增子。

It is not necessary to keep PCR tubes on ice as nonspecific DNA synthesis cannot occur at room temperature due to the inactive state of the of AllTaq DNA Polymerase.
由于 AllTaq DNA 聚合酶处于非活性状态，在室温下无法进行非特异性 DNA 合成，因此无需将 PCR 管置于冰上。

AllTaq PCR Kits are designed to be used with a final concentration of 0.25 μM.
AllTaq PCR 试剂盒的设计使用浓度为 0.25 μM。

The AllTaq Master Mix Kit can be used with genomic DNA, cDNA, plasmid DNA, oligonucleotides and other DNA molecules as template.
AllTaq Master Mix Kit 可以基因组 DNA、cDNA、质粒 DNA、寡核苷酸和其他 DNA 分子为模板。

Protocol
规程

You will set have 4 samples (“-“ and “+” for COL1A2 and “-“ and “+” for TBP). For each sample, label a 0.2ml PCR tube plus 2 tubes for a sample COL1A2 primers without template and TBP primers without template as negative controls.
您将获得 4 个样品（COL1A2 为"-"和 "+"，TBP 为"-"和 "+"）。对于每个样品，标记一个 0.2 毫升的 PCR 管，外加 2 个样品管，分别标记不含模板的 COL1A2 引物和不含模板的 TBP 引物作为阴性对照。

Prepare the reactions according to the table below. All Taq Master Mix contains PCR buffer,
根据下表准备反应。所有 Taq Master 混合液都含有 PCR 缓冲液、

dNTPs and AllTaq DNA polymerase.
dNTPs 和 AllTaq DNA 聚合酶。

Note: It is not necessary to keep samples on ice during reaction setup or while programming the cycler.
注意：在反应设置或循环仪编程时，无需将样品放在冰上。

Reaction setup
反应装置

Mix the reaction mixes gently but thoroughly, for example, by pipetting up and down a few times or vortexing for a few seconds. Make sure after mixing all the solution is in the bottom of the tube (not on the lid).
轻轻但彻底地混合反应混合物，例如，用移液管上下移动几次或涡旋几秒钟。确保混合后所有溶液都在试管底部（而不是盖子上）。

4. Program the thermal cycler using the conditions outlined in the table below:
4.使用下表中列出的条件对热循环仪进行编程：

Cycling conditions
骑行条件

Place the PCR tubes or plates in the thermal cycler and start the PCR program.
将 PCR 管或板放入热循环仪中，启动 PCR 程序。

At the end of the program, keep at 4^oC until collection.
程序结束时，保持在 4 ^o C，直到收集完毕。

Note: After amplification, samples can be stored at –20°C for longer storage.
注：扩增后，样本可在 -20°C 下保存更长时间。

For more information: AllTaq PCR Handbook www.qiagen.com/HB-2481
更多信息：AllTaq PCR 手册 www.qiagen.com/HB-2481

The basic melting temperature ™ of primers can be calculated as follows (Wallace rule):
引物的基本熔化温度 ™ 可按下式计算（华莱士法则）：

Tm = 2(A+T) + 4(G+C)

Where A, G, C, and T are the number of each nucleotide.
其中，A、G、C 和 T 是每个核苷酸的编号。

For example, Tm for the sequence TGCTCA is, 2(1+2) + 4(1+2) = 18°C.
例如，序列 TGCTCA 的 Tm 为 2(1+2) + 4(1+2) = 18°C。

Tm can also be calculated using an on-line calculator: http://tmcalculator.neb.com/#!/
也可使用在线计算器计算 Tm：http://tmcalculator.neb.com/#!/

Protocol 8: Agarose gel electrophoresis
规程 8：琼脂糖凝胶电泳

Agarose gel electrophoresis is used to separate nucleic acids according to their size. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. Depending on the size of the DNA to be visualised the percentage of agarose varies between 0.8% (for large DNA pieces) and 3% (for small DNA pieces). Low percentage agarose gels can be difficult to handle while high percentage agarose gels can be brittle and may not set evenly. Commonly 1-1.5% agarose is used.
琼脂糖凝胶电泳用于根据核酸的大小进行分离。较短的分子比较长的分子移动得更快，迁移得更远，因为较短的分子更容易通过凝胶的孔隙。根据待观察 DNA 的大小，琼脂糖的比例在 0.8%（大 DNA 片段）和 3%（小 DNA 片段）之间。低比例的琼脂糖凝胶可能难以处理，而高比例的琼脂糖凝胶可能会变脆且凝固不均匀。通常使用 1-1.5% 的琼脂糖。

To visualize DNA in the gel a dye is used. Previously highly toxic ethidium bromide (EtBr) was commonly used however this has been replaced by sensitive, stable and environmentally safe fluorescent nucleic acid dyes for staining dsDNA, ssDNA or RNA in agarose gels or polyacrylamide gels. Common dyes are SYBR^TMSafe (Invitrogen Catalogue number S33102) and GelRed® (Merck Catalogue number SCT122). When bound to the DNA the dyes fluorescence when exposed to blue light or UV excitation (470-530nm).
为了使凝胶中的 DNA 可视化，需要使用染料。以前常用的是剧毒的溴化乙锭（EtBr），但现在已被灵敏、稳定、对环境安全的荧光核酸染料所取代，用于染色琼脂糖凝胶或聚丙烯酰胺凝胶中的 dsDNA、ssDNA 或 RNA。常用的染料有 SYBR ^TM Safe（Invitrogen 公司产品目录编号 S33102）和 GelRed®（默克公司产品目录编号 SCT122）。染料与 DNA 结合后，在蓝光或紫外线激发下（470-530nm）会发出荧光。

It is important to be able to estimate the size of the DNA in experimental samples so a “ladder” of known DNA sizes (DNA Ladder) is run alongside the experimental samples for comparison. Examples of DNA ladders:
估算实验样本中 DNA 的大小非常重要，因此需要将已知 DNA 大小的 "梯子"（DNA 梯子）与实验样本一起进行比较。DNA 梯子的例子：

For a 1.5% agarose gel:
对于 1.5% 琼脂糖凝胶：

A solution of 1.5% agarose (molecular biology grade) in 1X Tris borate EDTA (TBE) buffer (supplied as 50X concentrated and diluted in distilled water) was prepared by gently heating 1.5g molecular biology grade agarose per 100mls of TBE in a microwave until the agarose has completely dissolved. Note: Tris acetate EDTA (TAE) can also be used as an alternative to TBE.
1 倍硼酸三乙二胺四乙酸（TBE）缓冲液（以 50 倍浓缩液供应，并用蒸馏水稀释）中的 1.5%琼脂糖（分子生物学级）溶液是在微波炉中轻轻加热每 100 毫升 TBE 中的 1.5 克分子生物学级琼脂糖，直到琼脂糖完全溶解。注：乙酸三乙二胺四乙酸（TAE）也可替代 TBE。

GelRed® a sensitive, stable and environmentally safe fluorescent nucleic acid dye designed to replace the highly toxic ethidium bromide (EtBr) for staining dsDNA, ssDNA or RNA in agarose gels or polyacrylamide gels was added to the molten agarose and mixed (10,000X stock solution diluted to 1X solution). The stain allows visualization of the PCR products under UV light.
GelRed® 是一种灵敏、稳定且对环境安全的荧光核酸染料，可取代剧毒的溴化乙锭（EtBr），用于染色琼脂糖凝胶或聚丙烯酰胺凝胶中的 dsDNA、ssDNA 或 RNA。染色剂可使 PCR 产物在紫外光下可视化。

The solution was aliquoted to ~60ml volumes of gel solution and kept warm to prevent the gel setting.
将溶液等分至 ~60 毫升凝胶溶液中，并保温以防止凝胶凝固。

Collect a bottle of gel solution from the oven.
从烤箱中收集一瓶凝胶溶液。

Ensure the gel is well mixed by gently swirling the bottle be careful to close the lid properly, the solution will be hot) and then pour the melted agarose into the sealed gel mould with comb in place. Make sure the gel is on a flat surface and free of bubbles (remove/pop bubbles with a pipette tip of necessary). Let the gel solidify for ~30 minutes before loading your samples. Note: If you are in a hurry the gel can be placed in a cold room.
轻轻旋转瓶子，确保凝胶充分混合（注意盖好盖子，溶液会很热），然后将融化的琼脂糖倒入密封的凝胶模具中，并用梳子梳好。确保凝胶表面平整无气泡（必要时用移液管吸头去除/挤出气泡）。让凝胶凝固约 30 分钟后再装载样品。注：如果时间仓促，可将凝胶置于冷藏室中。

Remove dam at either end of the gel and the comb.
清除凝胶和梳子两端的水坝。

Cover the gel with 1X TBE buffer.
用 1X TBE 缓冲液覆盖凝胶。

While the gel is setting prepare your samples and the DNA ladder. Label tubes and aliquot samples:
凝胶凝固时，准备好样品和 DNA 梯形图。给试管贴上标签并等分样品：

DNA ladder 2m
DNA 梯子 2ml

6X TriTrack DNA loading dye 2m
6X TriTrack DNA 上载染料 2ml

Water 8m
水 8ml

12ml

Sample (4 samples) 15m
样品（4 个样品） 15ml

6X TriTrack DNA loading dye 3m
6X TriTrack DNA 上载染料 3ml

18ml

(6X TriTrack DNA loading dye: 10mM TriHCl ph7.6, 0.03% bromophenol blue, 0.03% xylene cyanol FF, 0.15% orange G, 60% glycerol, 60mM EDTA)
(6X TriTrack DNA 上载染料：10mM TriHCl ph7.6、0.03% 溴酚蓝、0.03% 二甲苯青酚 FF、0.15% 橙 G、60% 甘油、60mM EDTA）。

In TBE the dyes run at the following equivalent sizes:
在三丁基环氧乙烷中，染料以下列等效尺寸运行：

Xylene cyanol 3,030 bp
二甲苯氰醇 3,030 bp

Bromophenol blue 220bp
溴酚蓝 220bp

Orange G <50bp
橙色 G <50bp

The dyes allow you to monitor electrophoresis to ensure the samples do not run off the gel.
通过染料，您可以监控电泳过程，确保样品不会从凝胶中流出。

Load samples into individual wells in the following order: DNA ladder – Sample 1, Sample 2, etc. Make a note of the location of your samples.
按以下顺序将样品装入各个孔中：DNA 梯形图 - 样品 1、样品 2 等。记下样品的位置。

Connect the gel unit to the power pack and run the gel at 100V for 30 – 60 minutes, depending on the size of the fragments and the separation you need to observe. The OrangeG band should be clearly visible ~1 cm from the bottom of the gel
将凝胶装置与电源箱连接，根据片段的大小和需要观察的分离情况，在 100V 电压下运行凝胶 30-60 分钟。在距凝胶底部 ~1 厘米处应能清晰看到 OrangeG 带。

Switch off the power supply and place the gel on the UVP gel documentation system to visualize the DNA band(s), take a picture of the gel.
关闭电源，将凝胶放在 UVP 凝胶记录系统上以观察 DNA 条带，并给凝胶拍照。

Estimate the size of the DNA fragment(s) obtained.
估计获得的 DNA 片段的大小。

Protocol 9: Enzyme-linked Immunoabsorbent Assay (ELISA)
规程 9：酶联免疫吸附试验（ELISA）

Quantitation of proteins (eg. insulin) is crucial in biomedical research. High specificity of antibodies for a particular protein provides a means for measuring the concentration that protein. Immunoassay is a generic term for all methods that use antibody-antigen interaction for quantification of a protein.
蛋白质（如胰岛素）的定量在生物医学研究中至关重要。针对特定蛋白质的高特异性抗体为测量该蛋白质的浓度提供了一种方法。免疫测定是利用抗体与抗原相互作用来定量蛋白质的所有方法的总称。

Enzyme-linked immunosorbent assay (ELISA). One of the commonest immunoassay techniques used in clinical and research laboratories is an ELISA. This method enables us to measure either an antigen (eg. insulin) or antibody (eg. anti-HIV antibody) in biological fluids.
酶联免疫吸附试验（ELISA）。酶联免疫吸附试验是临床和研究实验室最常用的免疫测定技术之一。通过这种方法，我们可以测量生物液体中的抗原（如胰岛素）或抗体（如抗艾滋病毒抗体）。

In this practical we will measure the concentration of insulin in samples of human serum using a commercial ELISA.
在本实践中，我们将使用一种商业 ELISA 方法来测量人体血清样本中的胰岛素浓度。

Principle of the Method:
方法的原理：

A known quantity of antibody is bound to the surface (plate).
已知数量的抗体与表面（平板）结合。

The antigen-containing sample is applied to the plate.
将含有抗原的样本涂抹到平板上。

The plate is washed to remove unbound antigen.
清洗平板以去除未结合的抗原。

A specific antibody is added which binds to the antigen. The antibody is conjugated to an enzyme eg. horseradish peroxidase (HRP).
加入与抗原结合的特异性抗体。抗体与一种酶结合，如辣根过氧化物酶（HRP）。

The plate is washed again to remove the unbound antibody-enzyme conjugates.
再次清洗平板，去除未结合的抗体-酶结合物。

A chemical is added which is converted by the enzyme into a colorimetric signal.
加入一种化学物质，由酶转化为比色信号。

The intensity (absorbance of the signal) is measured on spectrophotometer.
用分光光度计测量信号的强度（吸光度）。

Since spectrophotometers/plate readers measure the absorbance of the sample at a given wavelength, we need to calculate the concentration based on absorption of the sample. A standard curve is used to calculate concentration. A standard curve is a graph that indicates the relationship between concentration and absorption. Multiple standards with known concentrations are measured and plotted, which then allows the concentration of unknown samples to be determined by interpolation on the graph.
由于分光光度计/读板机测量的是特定波长下样品的吸光度，因此我们需要根据样品的吸光度来计算浓度。标准曲线用于计算浓度。标准曲线是表示浓度与吸收率之间关系的图表。测量并绘制多个已知浓度的标准曲线，然后就可以通过在曲线图上插值来确定未知样品的浓度。

Protocol
规程

Unknown samples are compared to dilutions of a standard with a known concentration of the antigen (insulin).
将未知样本与已知抗原（胰岛素）浓度的标准稀释液进行比较。

Pipette 50µl of each sample and standard into the appropriate well as shown:
如图所示，移取 50µl 样品和标准品到相应的孔中：