MEDC0066

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

(PLRS)
(PLRS) 翻译文本：

Protocol Manual 2024-25
协议手册 2024-25

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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.
课程将由诺曼教授、多诺万博士、普拉特博士以及研究生教学助理（PGTAs）授课，除非另有说明。

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协议检查页码！

APPENDICES
附件

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，你需要确保你了解你计划在实验室进行的任何活动的潜在危害。

• 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 已实施政策，确保员工、学生和访客在其工作场所安全，这些政策与 1974 年《工作场所健康与安全法》（HASAWA）一致。

GENERAL SAFETY MEASURES
一般安全措施

NO eating or drinking in the laboratory (including chewing gum and drinking water!).
实验室内禁止进食或饮水（包括嚼口香糖和饮水！）。

Make sure you know where to dispose of waste materials (see Waste Management, below).
确保您知道如何处理废弃物（见下文废物管理）。

Wash your hands before leaving the laboratory.
洗手后再离开实验室。

PERSONAL PROTECTIVE EQUIPMENT (PPE)
个人防护装备（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.
实验室内必须穿着实验服。实验服必须是高领、针织袖口和按扣式（豪伊式）闭合的，并且必须正确扣好。

All jewelry, scarves, ties, accessories etc that might become contaminated or cause entanglement should be contained within the lab coat.
所有珠宝、围巾、领带、配饰等可能被污染或引起缠绕的物品应存放在实验服内。

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.
在实验室必须佩戴眼罩，即使您佩戴眼镜进行视力矫正也不例外。

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.
尽管鞋子和适当的衣物不被视为个人防护装备，但它们必须为皮肤和双脚提供最低限度的保护。

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.
鞋类应完全封闭并覆盖双脚，必须能够抵抗有害物质或至少减缓双脚对有害物质的暴露。

Clothing should cover the legs to reduce skin exposure to splashes and spills.
衣物应覆盖腿部以减少皮肤暴露于溅洒和溢出物。

Gloves
手套

Where appropriate, wear gloves.
适当的时候，请戴手套。

Note: Gloves must be removed before touching e.g. door handles, lift call buttons or YOUR PHONE
注意：触摸例如门把手、电梯呼叫按钮或您的手机之前必须摘下手套

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包括以下几种：

Non-hazardous domestic waste (e.g. paper, packaging) – Clear or black bags
非危险生活垃圾（例如纸张、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（例如临床样本、细胞培养容器、手套）——黄色袋子、虎纹袋子（条纹）或高压蒸汽灭菌废物袋

Sharps (e.g. needles, scalpel blades, broken glass) – Yellow plastic sharps bins
锐器（例如针头、手术刀片、破碎玻璃）- 黄色塑料锐器容器

Pipets and pipet tips – Yellow and green bio-bins (bench or floor standing)
移液管和移液头 - 黄色和绿色生物垃圾桶（台式或立式）

Other: Ask the lab manager or the Health and Safety Officer.
其他：询问实验室经理或健康安全官员。

ACCIDENTS
意外事故

Accidents do happen in laboratories.
实验室里确实会发生事故。

Accidents can be due to human error, faulty equipment or inadequate training.
事故可能由人为错误、设备故障或培训不足引起。

ALL accidents should be reported to a member of staff.
所有事故都应报告给工作人员。

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.
为了降低损害/受伤的风险，在进行任何活动之前应进行风险评估。风险评估是一种旨在帮助识别与工作场所活动相关的危害、评估危害的潜在有害后果以及寻找避免或减少危害的方法的结构化过程。该过程最终产生一份书面安全作业系统，旨在确保风险降低到尽可能低的水平。

Definitions:
定义：

• Hazard: A property of a substance, activity or situation that has the potential to cause harm.
• 危害：物质、活动或情况具有潜在造成伤害的性质。

• Risk: The probability that the harm linked to a hazard will actually occur, taking into account also the severity of that harm.
• 风险：考虑到危害相关损害的严重性，该损害实际发生的概率。

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.
阅读以下场景，并记录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。他们在一个晚上在家做意大利面，第二天把剩下的意大利面带到实验室当午餐。实验室里有冰箱，所以他们把午餐放在冰箱里保持冷却。学生正在进行一个项目，分析患有肝病患者的样本。他们收集了一份人体肝脏样本，并将样本分成两半：他们将其中一半样本在液氮（-196°C）中冷冻以备将来分析，另一半用于酸消化。酸消化涉及将样本在浓盐酸（12M HCl）中孵育数小时。实验结束后，学生将手套扔进回收桶，洗手，从冰箱里拿出他们的午餐盒，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.
场景 2：一名学生加入了研究实验室进行项目。实验很有趣但耗时，他们从 9:00 开始，直到 22:30 才结束，学生必须独自留在实验室以确保所有提取柱在实验过程中正常工作。学生没有咖啡就无法工作，所以他们从星巴克买咖啡，在实验室做实验时喝。学生使用一种称为放射免疫测定的技术，该技术涉及使用放射性标签标记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并回家。

ESSENTIAL LABORATORY SKILLS
基本实验室技能

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.
制作溶液是实验室中最常见的活动之一。能够快速准确地制备复杂溶液是一项基本技能。

Work through the following problems (please show ALL your working/calculations in your lab book):
解决以下问题（请在您的实验记录本中展示所有工作/计算过程）：

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

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

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) 您需要制备 50ml 的溶液，最终浓度为：0.2%乙醇，5mM EDTA，20mM NaCl。试管中已含有 5ml 2%（体积比）乙醇和 5ml 50mM EDTA 水溶液。

You have a stock solution of 0.5M NaCl.
您有一瓶 0.5M 的氯化钠储备液。

What volume of 0.5M NaCl you need to add to the tube?
您需要向试管中加入多少毫升 0.5M 氯化钠？

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

c) You have stock solutions of:
c) 您有以下储备溶液：

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.
计算制备含有 0.2% SDS、0.1M EDTA、0.3M Tris 的 50ml 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 的 NaC 溶液。您需要制备 10 毫升 0.1mM 的 NaCl 溶液。您如何进行稀释？请写出所有步骤。

e) Tube A contains a dye called Trypan Blue.
e) 管 A 含有一种名为锥虫蓝的染料。

You need to dilute this by 10,000x to give 1ml of the final solution.
您需要将此溶液稀释 10,000 倍以得到 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.
列出使用水进行连续稀释的所有步骤，并制备 1 毫升的最终溶液。

2. BASIC LAB EQUIPMENT
2. 基本实验室设备

Each apparatus will be demonstrated by a member of staff.
每台设备将由一名员工演示。

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.
您有可供选择的：0.5ml、1.5ml、15ml 和 50ml 的试管以及 5ml、10ml 和 25ml 的移液管。选择能够最精确测量所需体积的移液管和可以容纳您所测量体积但不是过大不必要的单个试管。

Pipette the following volumes:
移液以下体积：

1.2ml, 2.5ml, 5ml

3.3ml, 5.4ml, 7.5 ml
3.3ml，5.4ml，7.5ml

11ml, 17ml, 21ml
11ml，17ml，21ml

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。体积相同吗？

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液到单独的 1.5 ml 微量离心管中：

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

P200 (yellow tips) – 130ml, 40ml, 25m
P200（黄色尖端）- 130ml，40m，25ml

P20 (yellow tips) – 19ml, 10ml, 3m
P20（黄色尖端）- 19ml，10m，3ml

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

ii) Balance
ii) 平衡

Task weigh a known volume of NaCl:
称取已知体积的 NaCl：

Select a weighboat
选择一艘衡船

Zero (Tare) the balance
零（去皮）称重

Weigh out 750mg NaC
称取 750mg NaCl

Make sure the balance is clean when you have finished.
确保完成时平衡是干净的。

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.
制备缓冲液需要一种快速准确测量溶液 pH 的方法。pH 计是一种用于测量溶液 pH 的设备。典型的 pH 计由一个特殊的测量电极（带有玻璃泡）组成，该电极连接到一个电子计，用于测量并显示 pH 读数。

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

Task measure to pH of two different solutions:
任务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.
请注意两种溶液的 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.
离心是利用离心力分离混合物的机械过程。它是实验室中最广泛使用的方法之一。在常规方案中，混合物被分离成两个部分——管底含有在施加的离心力下沉降的材料的沉淀物，以及含有未沉降材料的液体上清液。

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.
离心机有两个主要元件：一个转子用于固定待离心样品，一个电机用于产生力量。转子位于室内，以防在离心过程中发生故障时损坏。

Units used in centrifugation:
离心机使用的单位：

• RPM (Revolutions per minute): RPM is simply the speed of the motor.
• RPM（每分钟转数）：RPM 只是电动机的速度。

• 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 通常以重力加速度（g）为基准表示。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 依赖于转子的速度（RPM）以及半径。RCF 可以通过以下公式确定：

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.
注意：如果不知道转子的半径，则需要测量。半径是从转子中心到样本管底部的点测量的。

Or, by using a nomogram (overleaf) to find the RCF that is generated for a given RPM.
或者，使用（上页）的诺模图来查找给定 RPM 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）标尺计算转子速度以产生 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.
注意：在描述离心条件时，例如在论文或论文的“方法”部分，最好使用 RCF，因为这保持不变，而生成特定 RCF 所需的 RPM 则根据所使用的离心机和转子而变化。

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

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（转子中相对对角放置重量相等的试管）。

Calculate the rotor speed (RPM) to produce 1500 x g RCF
计算产生 1500 x g RCF 的转子速度（RPM）

Program the centrifuge to spin for 2 mins at 1500 x g
程序离心机旋转 2 分钟，转速 1500 x g

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!
在会议结束时，确保你的实验台整洁，所有废弃物都已正确放入废物容器中。保持实验台整洁是实验室必备的技能！

You must complete the tasks and have them signed off before leaving the class.
您必须完成任务并在离开课堂前得到签字确认。

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，涉及两种染料，苏木精染细胞核呈深蓝紫色，伊红染细胞质、细胞外基质和其他蛋白质结构呈粉红色。H&E 提供了细胞、组织和器官的微观解剖的全面图像。它可以用于染色石蜡包埋的组织切片、冷冻切片和细胞涂片。

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
补水

Haematoxylin
苏木精

Differentiation
区分

Bluing
蓝染

Eosin
嗜酸性细胞

Dehydration
脱水

Clearing
清除

Cover-slipping
盖玻片

Equipment
设备

Coplin jars
科普林瓶

Pencil
铅笔

Staining trays
染色盘

Forceps
镊子

Pastettes
过敏贴

Wash bottle
洗瓶

Liquid waste container
液体废物容器

Slides with tissue sections
组织切片幻灯片

Coverslips
盖玻片

Timer
计时器

Paper towels
卫生纸

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.
切片经 3 次（每次 5 分钟、2 分钟、1 分钟）在 Histoclear 或二甲苯中脱蜡，并通过在分级乙醇中浸泡进行复水：100%（3 分钟）、95%（2 分钟）和 70%（2 分钟），最后浸泡于水中。

Collect slides (2 slides labelled A and B) from the fume hood in a Coplin jar containing water.  
从烟罩中收集（标记为 A 和 B 的 2 张）载有水的 Coplin 瓶中的玻片。

Rinse once with deionized water to remove residual ethanol. 
用去离子水冲洗一次以去除残留乙醇。

Drain the slides well before staining (touch the corner of the slide to a paper towel to draw excess water off).
在染色前充分排干载玻片（将载玻片的角落接触纸巾以吸去多余水分）。

Label slides with your initials using a pencil and writing in the white/frosted area at the end of the slide.
在幻灯片末尾的白色/磨砂区域用铅笔标注您的姓名首字母。

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. 
将切片面朝上放置在染色架上，将染色皿中的足量过滤苏木精（H）均匀覆盖切片，孵育 5 分钟。

Wash with water and drain.  
用清水冲洗并排水。

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). 
将足够的蓝染试剂（BR）涂覆在切片上，使其完全覆盖，孵育 20 秒以区分染色（直到苏木精仅染色细胞核）。

Wash with water and drain as above.
用上述方法用水冲洗并排水。

Apply enough eosin (E) to completely cover the section and incubate for 4 min.
将足够的伊红（E）涂布在切片上，使其完全覆盖，孵育 4 分钟。

Wash away the stain with water and drain.  
用温水洗净污渍并排水。

Dehydrate sections by incubation in graded ethanols: 70% (1 min), 95% (1 min), 100% (2 min). 
通过分级乙醇（70%（1 分钟）、95%（1 分钟）、100%（2 分钟））孵育脱水切片。

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 3 次二甲苯中孵育（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.
将切片用盖玻片固定 - 在载玻片边缘滴一滴封片剂，轻轻放下盖玻片以将封片剂 spread over the section，注意避免气泡。晾干。

Observe the slides on a light microscope
观察载玻片在光学显微镜下

TIPS ON USING THE MICROSCOPE:
显微镜使用技巧：

• Always use the lowest power/magnification to find the focus and then work your way up to higher power.
• 总是使用最低的功率/放大倍数找到焦点，然后逐步提高功率。

• 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.
• 从粗焦点开始，一旦你能看到图像，就用细焦点旋钮进行微调。一旦一张幻灯片已经聚焦，你可能发现你可以更换幻灯片而无需从头开始。

• 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).
尽管只看一个目镜很诱人，但一开始用双眼聚焦比较困难，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:
对于每一张幻灯片，回答以下问题，并在实验记录本中记录你的答案：

Has the staining worked ie. purple nuclei, pink cytoplasm?
染色是否成功？即紫色核，粉红色细胞质？

Is there clear distinction between the two stains?
有沒有這兩種染料的明確區分？

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

What structures can you see?
您能看到哪些结构？

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

What differences are observed between the two slides?
观察到两张玻片之间有哪些差异？

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:
有各种软件可供分析科学图像，具体取决于您使用的显微镜和您希望进行的分析类型。一种非常广泛使用且公开可用的软件是 ImageJ：

https://imagej.net/software/imagej2

Protocol 2: Introduction to tissue culture, thawing cells from cryogenic stocks
协议 2：组织培养简介，从冷冻库中解冻细胞

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:
细胞培养是指从生物体中取出细胞，并在有利于保持原始组织生理和生化特性的人工环境中进行维护/生长。我们试图模拟特定细胞类型的生理条件是很重要的。细胞培养是一种广泛使用的技术，为以下提供模型系统：

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

Modelling disease
建模疾病

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
药物筛选与开发

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

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.
培养细胞需要无菌环境和营养物质的供应以促进生长。环境在 pH 和温度方面也应保持稳定。已经开发出许多培养基，并已商业化生产。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，并且可能还含有促进特定细胞类型生长的特定因素。当所有成分都已知时，该培养基被称为定义培养基。许多培养基含有血清（通常是胎牛血清），这是一种复杂且可变的因素混合物，可增强细胞增殖。不同细胞类型的细胞条件各不相同，未能提供适当的条件可能会减缓生长甚至导致细胞死亡。

Example of a commonly used cell culture medium:
常见细胞培养培养基的例子：

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.
杜贝科改良的伊格尔培养基（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）。胎牛血清（FCS，有时也称为胎牛血清（FBS）），通常以 10-20% v/v 的比例添加，以提供生长因子和营养来源。抗生素（通常使用青霉素和链霉素，最终浓度分别为 100U/ml 和 0.1mg/ml）也被添加，有时还添加抗真菌剂（通常是两性霉素 B，最终浓度为 0.25mg/ml）。抗生素/抗真菌剂混合物可以商业购买，例如从 Sigma 购买，作为 100X 储备液。

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.
细胞培养器皿和消耗品：无菌一次性组织培养塑料器皿——培养器皿、移液管和试管。有多种器皿可供细胞生长，但最常用的是烧瓶、培养皿和多孔板。器皿的基材聚苯乙烯本身不足以使大多数细胞粘附，因此表面经过改性以增加电荷。所有细胞培养塑料器皿的供应商都会对组织培养器皿的表面进行处理以产生表面改性。

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 II 级生物安全柜）以提供无菌工作环境。注意：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
保育箱（37 ^o C，5-10% CO ₂ ）提供细胞生长的最佳条件

Waterbath (37^oC) for pre-warming solutions
水浴（37 ^o C）预加热溶液

Inverted light microscope
倒置式光学显微镜

Centrifuge for pelleting cells
离心机用于细胞沉淀

Tips for aseptic technique and working the hood
注意事项：无菌技术及操作风柜的技巧

Use standard personal protective equipment (PPE) - lab coat, gloves and eye protection, make sure long hair is tied back or covered.
使用标准个人防护装备（PPE）- 实验室外套、手套和眼保护，确保长发扎起或遮盖。

Make sure all the surface is free of clutter.
确保所有表面无杂物。

Clean the working area inside the hood with 70% ethanol (EtOH)/IMS.
清洁罩内工作区域，使用 70%乙醇（EtOH）/IMS。

Before transferring bottles into the hood, disinfect all bottles with 70% EtOH/IMS by spraying or wiping.
在将瓶子转移到罩子之前，用 70%的乙醇/IMS 通过喷洒或擦拭对所有瓶子进行消毒。

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台的左侧，消耗品和废弃的烧杯放在右侧，保持中央区域空旷以便工作（每个人都能找到最适合他们的设置）。

Change or clean gloves with 70% EtOH/IMS if they are taken out of the hood or you suspect they might be contaminated.
更换或清洁 70%乙醇/IMS 手套，如果它们被取出风罩或您怀疑它们可能被污染。

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

Slow movement near and within the hood helps maintain constant circular airflow.
缓慢移动靠近和位于罩子内部有助于保持恒定的环形气流。

To help prevent contamination, as much as possible avoid speaking, sneezing and coughing while working in the hood.
为帮助预防污染，尽可能在工作时避免在罩内说话、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.
将滴管尖端、废试剂和废培养基丢弃到罩内适当标签的烧杯中。将使用过的塑料器皿丢弃到罩旁指定的垃圾桶中。

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.
完成清洁后，清除罩子并使用 70%的乙醇/IMS 擦拭区域。适当处理废弃物。罩子应保持清洁，以便下一个人使用。

Making medium for cell culture
制备细胞培养介质

Medium components:
中等成分：

- Dulbecco’s Modified Eagle’s medium (DMEM)
- 杜尔贝科改良的伊格尔培养基（DMEM）

- Foetal calf serum (FCS)
胎牛血清（FCS）

-   Antibiotic-Antimycotic solution (100X stock: penicillin, streptomycin, amphotericin-B)
- 抗生素-抗真菌溶液（100 倍浓缩：青霉素、链霉素、两性霉素-B）

Calculate the volumes needed to make 50mls growth medium:
计算制备 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.
- 培养基制备。

- 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.
- 从冷冻库中分离细胞。细胞冷冻保存允许细胞库存长期（不确定）保存。细胞储存在液氮中。

Thaw cells from cryogenic stocks
解冻冷冻库存细胞

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，根据计算出的体积配制 50 毫升培养基。确保您的培养基管上清楚地标有您的姓名首字母、日期和内容，因为您需要这种培养基用于方案 3 和 4。

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

Carefully remove a vial from liquid nitrogen using the correct PPE as instructed.
小心使用规定的个人防护装备从液氮中取出安瓿瓶。

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).
将安瓿瓶在 37℃水浴中解冻，轻轻搅拌直至仅可见最后的小块冰（仅用手指尖握住瓶盖）。

Take the vial to the hood.
将试管带到通风橱。

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 5 毫升移液器，从您的烧瓶中取出 1 毫升培养基，打开冷冻管，轻轻将细胞吸入移液器中的液体中。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.
将所有液体从移液管倒入烧杯中，并使用移液管轻轻混合细胞和培养基，上下轻轻吸取以避免产生气泡。

Label the flask with your name, the date, the name of the cells (copy what is written on the side of the cryovial).
贴上瓶子上的名字、日期、细胞名称（复制冻存管侧面的文字）。

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).
检查显微镜下的细胞，以确认细胞存在（细胞最初会漂浮，但应附着到基底上并扩散）。

Place the flask in the incubator making sure the flask is sitting flat on the shelf.
将烧瓶放入培养箱，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).
清理罩子 - 移除所有物品，用 70%乙醇（EtOH）或工业甲醇（IMS）擦拭消毒工作区域。

Store any solutions you will need for subsequent protocols. Cell culture medium is generally stored at 4^oC (in a fridge or cold-room).
储存您后续方案所需的任何溶液。细胞培养液通常储存在 4℃（冰箱或冷室中）。

Protocol 3: Subculture (passaging) of adherent cells and cell counting
协议 3：粘附细胞的传代（传代培养）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.
常规细胞亚培养对于维持培养、扩大细胞群体、为实验目的建立培养以及扩增用于冷冻保存的库存是必需的。进行亚培养和计数贴壁细胞时，必须将细胞从基质上分离并解离成单个细胞悬浮液。用于从基质上分离细胞的试剂有很多种，但常用的是胰蛋白酶/EDTA 混合物。胰蛋白酶是一种蛋白酶，EDTA 是一种螯合剂，它可以结合金属离子如钙和镁，这些离子是细胞粘附到基质和相互之间所必需的。在某些情况下，将酚红添加到胰蛋白酶/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.
一个可存活的细胞计数对于多种目的至关重要，例如细胞培养的管理、亚培养时细胞浓度的标准化、细胞产量的计算、诊断中细胞群体的滴定以及在工业生物过程中。细胞可以使用自动化细胞计数器或使用血细胞计数器手动计数。尽管细胞计数器快速、可靠且不易出错，但由于计数器和消耗品昂贵，手动使用血细胞计数器仍然因成本低廉和多功能性而被广泛使用。

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，在你的实验记录本上记录细胞形态——并评估细胞重叠程度（即细胞覆盖的表面积百分比）。

Take a photo of your cells.
拍摄您的细胞照片。

Cells are usually sub-cultured at around 80% confluency.
细胞通常在约 80%的汇合度时进行亚培养。

In the hood:
在罩壳内：

Aspirate medium from the flask taking care not to scratch the cell monolayer with the pipette.
吸取烧瓶中的培养基，注意不要用吸管刮伤细胞单层。

Wash cells with 10mls sterile Dulbecco’s modified phosphate-buffered saline (PBS) to remove residual serum which will neutralise protease activity.
用 10 毫升无菌的 Dulbecco 改良磷酸盐缓冲盐水（PBS）洗涤细胞，以去除残留的血清，防止其中和蛋白酶活性。

Aspirate all the PBS.
抽尽所有 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.
轻柔旋转以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℃孵育（最初 2 分钟，但时间会根据细胞类型和汇合程度而变化，每 2 分钟检查一次）。注意：细胞长时间暴露于胰蛋白酶-EDTA 中可能会损伤细胞。

Monitor detachment over time by checking under the microscope.
通过显微镜检查，监测随时间推移的脱落情况。

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.
一旦细胞脱落并形成单个细胞悬浮液（团块使细胞计数不准确），向其中加入 5 毫升生长培养基（来自方案 2）以中和蛋白酶。轻轻上下吹吸混合均匀。将细胞悬浮液倒入 15 毫升管中。

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).
细胞活力评估的锥蓝排除试验：染料排除试验用于确定细胞悬液中存活细胞的数量。该试验基于活细胞具有完整的细胞膜，可以排除某些染料，如锥蓝，而死亡细胞则不能。在此试验中，将细胞悬液与锥蓝简单混合，然后通过显微镜检查以确定细胞是否吸收染料（死亡细胞）或未吸收（存活细胞）。

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了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 ）的正方形组成。这九个正方形进一步被分成更小的正方形。大角正方形（进一步分成 16 个正方形）用于我们实验中的细胞计数。血细胞计数器的凸边将盖玻片保持在距标记网格 0.1 毫米的位置，为每个大正方形提供一个确定的体积。每个角正方形的体积为 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 细胞悬液至 1.5ml 微量离心管中。注意：取等份前充分混匀细胞悬液，以确保细胞分布均匀

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

Mix gently.
轻轻搅拌。

Load cells into the haemocytometer,10µl/chamber.
将负载细胞加入血细胞计数器，每室 10µl。

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非存活细胞的数量。通常计算 4 个角落的室。存活细胞明亮（因为它们排斥染料），死细胞暗淡（因为它们吸收染料）。在你的实验记录本上记录你的细胞计数。

Ideally ~100 cells/large square should be counted to increase the accuracy of the cell count.
理想情况下，应计数约 100 个细胞/大格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.
检查您的细胞计数与示范者一起，看看计数与他们的计数有多接近。

Calculate the concentration of viable and non-viable cells and the % of viable cells using the following equations where:
计算存活细胞和非存活细胞的浓度以及存活细胞百分比，使用以下公式，其中：

A = Mean number of viable cells counted
A = 平均可计数存活细胞数

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

B = Mean number of non-viable cells counted
B = 计算的非存活细胞平均数

Total number of non- viable cells counted/number of squares
总不可行细胞计数/方格数

C = Dilution factor
C = 稀释倍数

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 1mm ² ，腔体的深度为

0.1mm
0.1 毫米

The correction factor of 10⁴ converts 0.1mm³ to 1ml
10 ⁴ 的校正系数将 0.1mm ³ 转换为 1ml

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

Concentration of non-viable cells (cells/ml) = B x C x D
非存活细胞浓度（细胞/ml）= 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 = 存活细胞数 + 非存活细胞数

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

Possible sources of inaccuracy in the count:
可能的计数不准确来源：

Bubbles and debris in the chamber.
气泡和碎片在室内。

Overfilling the chamber such the sample runs into the channels or the other chamber
过充容器导致样本流入通道或另一个容器

Incomplete filling of the chamber.
不完全填充腔室。

Cells not evenly distributed throughout the chamber and/or not in a single cell suspension.
细胞在腔内分布不均匀，或不在单一细胞悬液中。

Too few cells to count. If necessary, this can be overcome by centrifuging the cells, resuspending in a smaller volume and recounting.
细胞数量过少，无法计数。如有必要，可以通过离心细胞，在更小的体积中重新悬浮并重新计数来克服这个问题。

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。这可以通过在 Trypan Blue 中使用更高的稀释倍数来克服，例如 1 体积细胞：10 体积 TB。

Passaging cells
通道细胞

Calculate the number of cells needed to plate all the wells of a 6-well plate at 0. 5x10⁶ cells/well.
计算需要多少细胞来接种 6 孔板的全部孔，浓度为 0.5x10^6 细胞/孔。

Note: In a 6-well plate each well contains 2ml of medium so you want 12mls at 0.25x10⁶ cells/ml.
注意：在 6 孔板中，每个孔含有 2 毫升培养基，因此您需要 12 毫升的 0.25x10 ⁶ 细胞/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，并将其分装到 15 毫升试管中（在取分装量之前确保通过反复倒置混合细胞悬浮液，因为细胞会随着时间的推移沉到试管底部，例如在计数细胞数量时）。

Make the volume up to 12ml total volume with growth medium.
将总体积调至 12ml，包括生长培养基。

Plate cells into a 6 well plate, 2ml/well.
将细胞接种于 6 孔板，每孔 2 毫升。

Label the plate with the cell type, date and your name.
标明板上的细胞类型、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。用肉眼观察，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.
将培养皿放入孵箱，确保培养皿平放在架子上。在 37℃、5%CO2 条件下培养。

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

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.
细胞培养常用于模拟疾病。例如，常用的纤维化疾病模型是对细胞进行已知致纤维化因子（如转化生长因子 b（TGF)b 或血小板源性生长因子（PDGF））的处理，并比较处理组与未处理（对照组）细胞的基因和蛋白质表达。注意：溶剂是指用于重新构成/稀释生长因子的溶液。对照组细胞用等体积的溶剂处理，以消除观察到的任何变化是由于溶剂而不是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 3 号方案中的 6 孔板细胞，您将处理一半的细胞（3 个孔）用 TGFb 处理，另一半用相同的中含有等体积载体的同种介质（载体处理/对照组）。对于特定生长因子的处理，介质中的 FCS 要么去除，要么严重减少，例如减少到 0.5-1%，因此可以研究特定因素的效果，而不受未定义血清因素的影响。

Prepare 50ml DMEM containing 0.5% FCS and 1% antibiotic/antimycotic as for Protocol 2:
准备含有 0.5% FCS 和 1%抗生素/抗真菌剂的 50ml DMEM，如协议 2 所述：

Prepare medium with and without TGFb 2ng/ml:
准备含/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 毫升培养基分装到两个 15 毫升试管中，标记试管为“TGFb”和“Vehicle”。

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.
注意：制备用于处理细胞的溶液时，建议多制备一些，以确保有足够的量来处理所有孔板，并使用完整体积。

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 TGFb 2ng/m 的储备液，浓度为 4mM HCl，1mg/ml 的牛血清白蛋白（BSA），以及 4mM HCl，1mg/ml BSA（溶剂）的储备液。

Calculate the volume of TGFb you will need to add to 7mls to give a final concentration of 2ng/ml.
计算需要添加到 7 毫升中以达到最终浓度为 2ng/ml 的 TGFb 的体积。

Add TGFb to the 7mls medium, close the tube tightly and mix by inverting gently.
将 TGFb 加入 7mls 培养基中，紧闭管口，轻轻倒置混匀。

Add an equivalent volume of vehicle to the second tube and mix.
向第二个试管中加入等量的车辆并混合。

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.
从孵箱中取出 6 孔板，在显微镜下检查细胞——记录形态和汇合度，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：在洗涤和处理细胞时，尽量保持培养皿覆盖，以最大限度地减少细胞干燥的机会。

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.
从孔中吸出培养基，加入 DMEM + 0.5% FSC+1%抗生素/抗真菌剂 2ml/孔，轻轻摇动板子以清洗细胞。

Aspirate the medium.
吸取培养基。

Add the appropriate medium to the wells, 2ml/well.
将适当的介质加入孔中，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.
标明板子以示，3 个孔“对照”或“-”，即无 TGFb，3 个孔“TGFb”或“+”，以便您知道每个孔中的细胞接受了什么处理。

Check the cells under the microscope (to ensure you still have healthy cells attached to the plate!).
检查显微镜下的细胞（以确保你仍然有健康的细胞附着在培养皿上！）。

Replace the plate in the incubator, again ensuring the plate is sitting flat on the shelf.
更换培养箱中的板，再次确保板子平放在架子上。

Harvesting cells for RNA and protein expression analyses
采集细胞进行 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.
在适当的孵育时间（通常为 24-72 小时）后，收集处理过载体的细胞和 TGFb 处理的细胞，以分析 RNA 和蛋白质表达。

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.
从培养箱中取出板，在显微镜下检查细胞，记录载药处理和 TGFb 处理细胞的形态和汇合度。两组之间有差异吗？您可能想拍照。

Place the 6-well plate on ice and remove the medium.
将 6 孔板放在冰上并移除培养基。

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

Leave the cells covered with ice-cold PBS to avoid cells drying out as wells will be processed at slightly different times.
将冰冷的 PBS 覆盖的细胞留置，以避免细胞干燥，因为细胞将在不同时间进行处理。

RNA sample preparation
RNA 样本制备

Efficient disruption and homogenisation of the starting material is essential for RNA purification.
高效地破坏和均质化起始材料对于 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.
细胞和细胞器细胞壁和质膜的完全破坏对于释放样本中所有 RNA 至关重要。不完全破坏会导致 RNA 产量显著降低。均质化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物的粘度，从而创建均匀的裂解物。不完全均质化会导致 RNA 与离心柱膜的结合效率低下，因此显著降低 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：在提取和处理 RNA 时，必须使用 RNA 酶无污染的溶液和设备，以最大限度地减少环境 RNA 酶对 RNA 的降解。RNA 酶的主要来源之一是您的双手，确保您戴上手套！

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 的 RNeasy Mini 试剂盒提取（见方案 5）。样品首先在含有高度变性胍硫氰酸盐的缓冲液中裂解和匀质化，该缓冲液可灭活任何 RNA 酶。

Tilt the plate and remove all the PBS from one “Vehicle” well and one “TGFb” well.
倾斜板，从“载体”孔和“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 600ml Buffer RLT（裂解缓冲液）并使用细胞刮刀将细胞从培养皿上刮下。

Transfer the lysate to a clean RNAse-free 1.5ml microcentrifuge tube and vortex.
将裂解物转移到无菌 RNA 酶-free 1.5ml 离心管中，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.
将裂解物使用 20 号（20G）针头连接的塑料注射器进行均质化。将裂解物轻轻通过针头 5-10 次，直至获得均质裂解物。注意：小心不要产生气泡。

Check by eye that the two samples have a similar volume.
检查两个样本的体积是否相似。

Store the lysate at -20^oC or -70^oC for subsequent use.
将裂解物储存在-20℃或-70℃以备后续使用。

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.
倾斜板至一定角度，并从“载体”孔和“TGFb”孔中移除所有 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，1X 蛋白酶和磷酸酶抑制剂）至细胞中，并使用细胞刮刀收获。

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.
将细胞裂解物转移到 1.5 毫升微量离心管中，在室温下以最大速度在微量离心机中离心 10 分钟，以沉淀任何不溶性物质。

Transfer the supernatant to a clean tube, taking care not to disrupt the pellet.
将上清液转移到干净试管中，注意不要破坏沉淀物。

Check by eye that the two samples have a similar volume.
检查两个样本的体积是否相似。

Store the supernatant at -20^oC for subsequent use. Discard the pellet.
将上清液保存在-20℃下备用。弃去沉淀物。

Samples can be stored at -70^oC for longer term storage.
样本可存储于-70℃以进行长期保存。

Protocol 5: Extraction of RNA from LX2 cells using the RNeasy Mini Kit
方案 5：使用 RNeasy Mini Kit 从 LX2 细胞中提取 RNA

(QIAGEN Catalogue number: 74104)
（QIAGEN 产品目录号：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 程序代表了一种成熟的 RNA 纯化技术。这项技术结合了基于硅酸盐膜的特异性结合特性和微旋技术的速度。一种特殊的高盐缓冲系统允许高达 100 µg 的 RNA 结合到 RNeasy 硅酸盐膜上。生物样本首先在含有高度去污的胍-硫氰酸盐缓冲液的存在下裂解和均质化，该缓冲液立即灭活 RNases 以确保纯化完整的 RNA。加入乙醇以提供适当的结合条件，然后将样本应用于 RNeasy Mini 旋柱，其中总 RNA 结合到膜上，污染物被有效洗去。然后将高质量的 RNA 在 30-100 µl 水中洗脱。使用 RNeasy 程序，所有大于 200 个核苷酸的 RNA 分子都被纯化。该程序对 mRNA 进行了富集，因为大多数小于 200 个核苷酸的 RNA（如 5.8S rRNA、5S rRNA 和 tRNA，它们共同占总 RNA 的 15-20%）被选择性排除。

Protocols for purification of RNA using RNeasy Kits are available at:
RNA 纯化使用 RNeasy 试剂盒的方案可在以下地址获取：

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:
流程图来自 Qiagen 手册的方案：

Protocol
方案

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).
以下方案适用于 6 孔板中 1 孔的汇合粘附细胞。每对成员可以做一个样本（1 孔）。

Make sure you know which sample is Vehicle and which is TGFb - MAKE SURE TUBES ARE LABELLED!
确保你知道哪个样本是车辆和哪个是 TGFb - 确保试管已标记！

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.
将协议 4 中的裂解物在室温下解冻，直至完全解冻且盐类溶解。避免长时间孵育，这可能会损害 RNA 的完整性。如果可见任何不溶性物质，则在微型离心机中以最大速度离心 5 分钟，并将上清液转移到新的 RNA 酶无菌管中。

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%乙醇，并通过吸管充分混合。

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 离心柱（粉色柱）放入 2 毫升收集管中，每个样本一个柱子。确保柱子已标记，以便知道哪个样本在哪个柱子中。将样本的一半（最大体积 700 毫升）转移到柱子上，注意不要触摸柱子底部的膜。将柱子/收集管放入离心机中，轻轻关闭盖子，以 9000 x g 的速度离心 15 秒。弃去流过物，即收集管中的溶液。重复此步骤，使整个样本通过柱子。弃去流过物。

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.
将 700µl 缓冲液 RW1 加入 RNeasy 离心柱。轻轻关闭离心机盖，以最大速度离心 15 秒以清洗离心柱膜。弃去流穿液。

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 离心柱中加入 500µl 缓冲液 RPE。轻轻盖上盖子，以最大速度离心 15 秒以清洗离心柱膜。弃去流穿液。

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.
向 RNeasy 离心柱中加入 500µl 缓冲液 RPE。轻轻盖上盖子，以最大速度离心 1 分钟。弃去流穿液。

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.
旋转 3 分钟以完全干燥膜。这个较长的离心步骤干燥了旋转柱膜，确保在洗脱步骤中不会携带乙醇。离心后，小心地将 RNeasy 旋转柱从收集管中取出，确保柱子不接触流动部分，以避免携带乙醇。

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 离心柱放入新的 1.5 ml 收集管中。直接将 30µl 无 RNA 酶水加到离心柱膜的中心。注意：不要用吸管尖端触摸膜。轻轻盖上盖子，在室温下静置 1 分钟，然后以全速离心 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.
使用少量 RNA（1-2 毫升）来测量浓度（见下文）。剩余部分应存放在冰上以备立即使用，或为长期储存，存放在-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 浓度和纯度使用名为 Nanodrop（赛默飞世尔）的微体积分光光度计进行评估，该仪器显示样品的吸光度谱。确保您记录这些值。

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.
RNA 在纯样品中的浓度可以使用分光光度计进行测量。下图中显示 260 nm 处的最大吸收。因此，可以通过测量稀释液在 260nm 处的吸光度（A260）来确定 RNA 的浓度。为确保样品纯净，可以在两个不同的波长下测量吸收，并将比值与纯 RNA 样品预期的吸收比值进行比较。RNA 的纯度由 A260/A280 和 A260/A230（A=吸光度）的比值给出。

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 低于预期通常表明蛋白质污染。

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

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，这是一种基于不同波长的吸光度来给出 RNA、DNA 和蛋白质吸光度谱的光谱光度计。首先将为您演示该仪器的使用，然后您可以分析您的样品。

Using a Nanodrop Spectrophotometer:
使用纳米滴定分光光度计：

1. From the home screen, select the Nucleic Acid tab and tap “RNA”.
1. 从主页选择核酸标签，点击“RNA”。

2. To blank the machine, pipette 2µl of RNase-free water onto the lower pedestal and lower the arm.
2. 为清空机器，将 2µ的 RNase-free 水滴入下方的底座，并降低臂部。

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

4. Lift the arm and clean both pedestals with a clean laboratory wipe.
4. 抬起手臂，用干净的实验室擦拭布清洁两个底座。

5. To measure samples, pipette 2µl of sample onto the pedestal and lower the arm, tap “Measure”.
5. 为测量样本，将 2µ的样本滴在底座上，降低臂部，轻触“测量”。

6. Clean the pedestal with a clean wipe and repeat step 5 for the next sample.
6. 用干净的布擦拭底座，并对下一个样本重复步骤 5。

7. Record your result in your lab book using the table shown below:
7. 使用下表记录您的实验结果到您的实验记录本中：

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

9. Lift the arm and clean the pedestal with a clean wipe.
9. 抬起手臂，用干净的布擦拭底座。

Make a note of your interpretation of what the concentrations and A260/A280 and A260/A230 ratios mean as they relate to your samples.
请注意您对样品中浓度以及 A260/A280 和 A260/A230 比值含义的解释。

If ratios are low, make a note in your lab-book of possible causes of reduced RNA purity?
如果比例低，请在您的实验记录本中记录可能降低 RNA 纯度的原因？

Protocol 6: Reverse transcription using QuantiTect Reverse Transcription Kit (QIAGEN Catalogue number: 205311)
协议 6：使用 QuantiTect 逆转录试剂盒（QIAGEN 产品目录号：205311）进行逆转录

Background
背景

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.
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. 定量 Tect 逆转录试剂盒专为两步 RT-PCR 使用而设计，并为低丰度转录本的敏感定量提供高 cDNA 产量。

The procedure comprises 2 main steps (see Protocol flowchart below):
该程序包括 2 个主要步骤（见下方的流程图协议）：

Elimination of genomic DNA
清除基因组 DNA

Genomic DNA contamination in RNA samples must be eliminated to avoid detection of genomic DNA rather than cDNA.
基因组 DNA 污染在 RNA 样本中必须被消除，以避免检测到基因组 DNA 而不是 cDNA。

Reverse transcription
逆转录

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.
在移除基因组 DNA 后，使用含有 Quantiscript 逆转录酶、Quantiscript RT 缓冲液和 RT 引物混合物的 Master Mix 对 RNA 样本进行逆转录。Quantiscript 逆转录酶对 RNA 具有高亲和力，并优化了从 10pg - 1μg 的 RNA 中高效且灵敏地合成 cDNA。高 RNA 亲和力，即使是从高 GC 含量或复杂二级结构的模板中，也能实现高 cDNA 产量。RT 引物混合物确保从 RNA 转录本的各个区域进行 cDNA 合成，即使是 5'区域。这允许无论目标区域在转录本中的位置如何，都能获得高产量 cDNA 模板，以便进行 PCR 分析。

Protocol
方案

QuantiTect Reverse Transcription Procedure
QuantiTect 逆转录步骤

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

Label a 1.5ml tube for each of your samples.
为每个样本贴上 1.5 毫升管的标签。

Calculate the volume of sample you need for 1mg RNA.
计算您需要的样本体积以获得 1mg RNA。

Mix and briefly centrifuge each component before use.
混合并使用前对每个成分进行离心分离。

Combine the following:
Combine the following: 合并以下内容：

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

Centrifuge briefly to remove any condensation from the lid of the tube.
离心管盖上的冷凝水，请短暂离心去除。

Prepare the following cDNA Synthesis mix, adding each component in the indicated order:
准备以下 cDNA 合成混合物，按照指示顺序添加每个成分：

.

Incubate as follows:
孵化方法如下：

 - 15 minutes at 42°C.
- 15 分钟，42°C。

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

- Chill on ice.
- 冰敷。

cDNA synthesis reaction can be used for PCR immediately or stored at -20°C for future use.
cDNA 合成反应可立即用于 PCR 或储存于-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 需要立即用于 PCR，就像这里的情况一样，用无 DNA 酶的水稀释至 1:10。

Protocol 7: Polymerase Chain Reaction using AllTaq DNA polymerase (Qiagen Catalogue number: 203144)
协议 7：使用 AllTaq DNA 聚合酶进行聚合酶链反应（Qiagen 产品目录号：203144）

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.
聚合酶链反应（PCR）是一种用于快速扩增特定 DNA 序列的技术。PCR 利用 DNA 聚合酶、引物和 dNTPs 在 DNA 模板上扩增序列。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)).
在纯化总 RNA 并将其逆转录为 cDNA 后，将进行 PCR 反应以研究 LX2 细胞中胶原蛋白 1（ECM 的主要成分）的表达，并确定其表达是否受 TGFb 在此细胞类型中的调控。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:
引物序列用于扩增胶原蛋白 1（COL1A2）转录本如下：

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.
作为阳性对照，也使用“管家”或组成型表达的基因。常用的基因是 TBP（TATA 框结合蛋白），这是一种通用转录因子，是真核转录机器的关键组成部分。

The sequence of the primers for amplification of the TBP transcript are:
引物序列用于扩增 TBP 转录本如下：

Notes
注意事項

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

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.
不需要将 PCR 管放在冰上，因为由于 AllTaq DNA 聚合酶的非活性状态，非特异性 DNA 合成在室温下无法发生。

AllTaq PCR Kits are designed to be used with a final concentration of 0.25 μM.
AllTaq PCR 试剂盒设计用于与 0.25 μM 的最终浓度一起使用。

The AllTaq Master Mix Kit can be used with genomic DNA, cDNA, plasmid DNA, oligonucleotides and other DNA molecules as template.
The AllTaq Master Mix Kit 可用于基因组 DNA、cDNA、质粒 DNA、寡核苷酸以及其他 DNA 分子作为模板。

Protocol
方案

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.
您将设置 4 个样本（COL1A2 的“-”和“+”，TBP 的“-”和“+”）。对于每个样本，标记一个 0.2ml 的 PCR 管，以及 2 个用于 COL1A2 引物无模板和 TBP 引物无模板的样本作为阴性对照管。

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 Mix 包含 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：在反应设置或编程循环器时，没有必要将样品保持在冰上。

Reaction setup
反应装置设置

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，通过上下吸管几次或涡旋几秒钟。确保混合后所有溶液都在管底（不在管盖上）。

4. Program the thermal cycler using the conditions outlined in the table below:
4. 使用下表中所列条件编程热循环仪：

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，并启动 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 直至收集。

Note: After amplification, samples can be stored at –20°C for longer storage.
注意：放大后，样本可存储于-20°C 以进行长期保存。

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):
基本熔解温度™的公式如下（Wallace 规则）：

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

Where A, G, C, and T are the number of each nucleotide.
A、G、C 和 T 分别代表每种核苷酸的数量。

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

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

Protocol 8: Agarose gel electrophoresis
协议 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。较短的分子移动更快，迁移距离比长的分子更远，因为较短的分子更容易通过凝胶的孔隙。根据要可视化的 DNA 大小，琼脂糖的百分比在 0.8%（用于大 DNA 片段）到 3%（用于小 DNA 片段）之间变化。低百分比琼脂糖凝胶可能难以处理，而高百分比琼脂糖凝胶可能脆弱且可能不均匀凝固。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).
为了在凝胶中可视化 DNA，使用染料。以前常用高度有毒的乙锭溴化物（EtBr），但现已由对 dsDNA、ssDNA 或 RNA 在琼脂糖凝胶或聚丙烯酰胺凝胶中染色的敏感、稳定且环保的荧光核酸染料所取代。常见的染料有 SYBR ^TM 安全（Invitrogen 产品目录号 S33102）和 GelRed®（Merck 产品目录号 SCT122）。当与 DNA 结合时，染料在蓝光或紫外激发（470-530nm）下发出荧光。

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:
重要的是能够估计实验样本中 DNA 的大小，因此需要将已知 DNA 大小的“梯子”（DNA Ladder）与实验样本一起运行以进行比较。DNA 梯子示例：

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

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.
1.5%琼脂糖（分子生物学级）的 1X Tris 硼酸 EDTA（TBE）缓冲液（以 50X 浓缩液形式提供，用蒸馏水稀释）通过在微波中轻轻加热每 100ml TBE 中的 1.5g 分子生物学级琼脂糖直至琼脂糖完全溶解来制备。注意：Tris 乙酸盐 EDTA（TAE）也可以作为 TBE 的替代品。

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®是一种敏感、稳定且环保的荧光核酸染料，旨在替代高度有毒的乙二胺溴化物（EtBr），用于在琼脂糖凝胶或聚丙烯酰胺凝胶中染色双链 DNA、单链 DNA 或 RNA。将其添加到熔融的琼脂糖中并混合（将 10,000X 浓缩液稀释至 1X 溶液）。该染料允许在紫外线下观察 PCR 产物。

The solution was aliquoted to ~60ml volumes of gel solution and kept warm to prevent the gel setting.
溶液被分装成约 60 毫升的凝胶溶液体积，并保持温暖以防止凝胶凝固。

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.
确保凝胶充分混合，轻轻摇匀瓶子，注意正确关闭盖子（溶液会变热），然后将融化的琼脂倒入装有梳子的密封凝胶模具中。确保凝胶放在平坦的表面上，无气泡（如有必要，用滴管尖端移除/弹出气泡）。在加载样品前，让凝胶凝固约 30 分钟。注意：如果您急于完成，可以将凝胶放入冷室。

Remove dam at either end of the gel and the comb.
移除凝胶两端和梳子的挡板。

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

While the gel is setting prepare your samples and the DNA ladder. Label tubes and aliquot samples:
当凝胶凝固时，准备您的样本和 DNA 梯子。标记试管并分装样本：

DNA ladder 2m
DNA 电泳梯子 2ml

6X TriTrack DNA loading dye 2m
6X TriTrack DNA 加样染料 2ml

Water 8m
水 8ml

12ml

Sample (4 samples) 15m
样本（4 个样本）15ml

6X TriTrack DNA loading dye 3m
6X TriTrack DNA 上样染料 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 染色剂：10mM TriHCl pH7.6，0.03%溴酚蓝，0.03%二甲苯氰 FF，0.15%橙 G，60%甘油，60mM EDTA)

In TBE the dyes run at the following equivalent sizes:
在 TBE 中，染料在以下等效尺寸下运行：

Xylene cyanol 3,030 bp
对苯二酚氰 3,030 bp

Bromophenol blue 220bp
溴酚蓝 220bp

Orange G <50bp
橙 G <50bp

The dyes allow you to monitor electrophoresis to ensure the samples do not run off the gel.
染料使您能够监控电泳，以确保样品不会从凝胶中跑出。

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.
将样本按照以下顺序装入单独的孔中：DNA 梯子 - 样本 1，样本 2，等等。记录下您样本的位置。

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
将凝胶单元连接到电源包，以 100V 电压运行凝胶 30-60 分钟，具体时间取决于片段大小和需要观察的分离程度。OrangeG 条带应在凝胶底部约 1 厘米处清晰可见

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，将凝胶放置在 UVP 凝胶成像系统上以可视化 DNA 条带，对凝胶拍照。

Estimate the size of the DNA fragment(s) obtained.
估算获得的 DNA 片段大小。

Protocol 9: Enzyme-linked Immunoabsorbent Assay (ELISA)
协议 9：酶联免疫吸附测定（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（例如胰岛素）的定量在生物医学研究中至关重要。针对特定蛋白质的抗体具有高特异性，这为测量该蛋白质的浓度提供了一种方法。免疫测定是所有使用抗体-抗原相互作用进行蛋白质定量方法的通用术语。

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.
酶联免疫吸附测定（ELISA）。在临床和研究实验室中，最常用的免疫测定技术之一是 ELISA。这种方法使我们能够测量生物流体中的抗原（例如胰岛素）或抗体（例如抗 HIV 抗体）。

In this practical we will measure the concentration of insulin in samples of human serum using a commercial ELISA.
在这个实验中，我们将使用商业 ELISA 测量人血清样本中胰岛素的浓度。

Principle of the Method:
方法原理：

A known quantity of antibody is bound to the surface (plate).
已知量的抗体结合到表面（板）。

The antigen-containing sample is applied to the plate.
抗原样本被应用于板子上。

The plate is washed to remove unbound antigen.
板被清洗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).
一种特定的抗体被添加，该抗体与抗原结合。该抗体被连接到一种酶上，例如辣根过氧化物酶（HRP）。

The plate is washed again to remove the unbound antibody-enzyme conjugates.
该板再次清洗以去除未结合的抗体-酶偶联物。

A chemical is added which is converted by the enzyme into a colorimetric signal.
一种化学物质被添加，该物质被酶转化为比色信号。

The intensity (absorbance of the signal) is measured on spectrophotometer.
信号强度（信号吸收度）在分光光度计上测量。