驶入细胞分析的高速路

驶入细胞分析的高速路

Promega细胞学产品介绍

驶入细胞分析的高速路

Promega细胞学产品介绍

付艳荣

市场销售部经理

客户的需求

• 研究对象－－细胞

是多种成分组成的复杂体，对任何一个参数的量化都是异常困难的。

• 想要准确定量细胞内众多的生理变化。

• 不需要过多地优化实验条件，争取一次成功。

• 需要 convenient, easy-to-use, off-the-shelf assays so personnel do not need to become experts

优秀的检测试剂盒能够带给你-

• Better Data

• More Publications

• More Grants

• More Time

• More Money

• Success!

Promega’s cell based assay products

Cell Viability / Cytotoxicity Assays

• CellTiter-Glo® Assay• CytoTox-ONE™ Assay • CellTiter-Blue® Assay• CellTiter 96® AQueous One

Solution • Live Dead Assay

Apoptosis Assays• Caspase-Glo 3/7® &

Caspase-Glo® 8 & 9 Assays• Apo-One® Caspase-3/7

Assay

Reporter Gene Assays• Dual-Glo® Luciferase Assay• Dual-Luciferase® Reporter

Assay• Chroma-Glo™ Luciferase

Assay• Bright-Glo™ and Steady-

Glo® Luciferase Assays• pGL4 Vector series• uHTS Dual Reagent

GloMax™ Luminometers

Promega细胞学产品介绍PromegaPromega细胞学产品介绍细胞学产品介绍

细胞活力检测

细胞凋亡检测

细胞信号转导

细胞活力检测

活细胞检测 – cell viability死细胞检测 - cytotoxicity

In Vitro Cell Viability/Cytotoxicity Assay Scheme

Equilibration Exposure Assay

SeedCells

Add TestCompound or Treatment

Add AssayReagent

RecordData

0-24 hrs 4 hrs – 5 days 10 min–24 hrs

Potential Outcomes v. ControlNo effect Proliferative Effect Cytotoxic Effects

- Apoptosis- 1° Necrosis

定义

• Viability = The quality or state of being alive. - Membrane integrity - Metabolic capacity

• Cytotoxicity = The quality or state of being deadly or poisonous to cells. – Necrosis– Apoptosis

活细胞检测

基于琥珀酸脱氢酶的检测– 酶标仪

基于其他氧化还原酶的检测– 荧光分光光度计

基于ATP的检测– 发光检测计

活细胞检测

基于琥珀酸脱氢酶的检测

基于其他氧化还原酶的检测基于其他氧化还原酶的检测基于其他氧化还原酶的检测

基于基于基于ATPATPATP的检测的检测的检测


四唑盐甲臢甲臢

琥珀酸脱氢酶

• CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) G4000 G4100

• CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) G5421 G5430 G5440

• CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) G3582 G3580 G3581

CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT)

• Dye solution

• Solubilization solution/Stop mix

试剂盒组分:

CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT)

加样 Dye Solution

37℃孵育4小时

加样 Solubilization/Stop Solution

比色 570nm

孵育1小时

前期培养

产品规格

• G4000 1000 assays =￥1926; ￥1.93 /well• G4100 5000 assays = ￥5436; ￥1.09 /well

CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS)

• MTS solution

• PMS solution

试剂盒组分:


混合MTS 、PMS

加样

37℃孵育1-4小时

比色 490nm

前期培养


• 易于使用混合 – 加样 – 孵育 – 读数

• 灵活可随时观察显色反应

• 快速无需洗涤细胞、无需溶解产物

• 安全无需刺激性有机溶剂SDS

• 方便现成的、稳定的、无菌的溶液

• 非放射性无需放射性同位素的操作资格及相应费用

产品规格

• G5421 1000 assays = ￥1647; ￥1.65 /well

• G5430 5000 assays = ￥5436; ￥1.09 /well

• G5440 50000 assays = ￥44793; ￥0.90 /well

CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)

• CellTiter 96® AQueous One Solution Reagent （MTS & PES）

试剂盒组分:

加样 …reagent

37℃孵育1-4小时

比色 490nm


前期培养

• 易于使用加样 – 孵育 – 读数

• 方便现成的、稳定的、无菌的单一溶液

• 快速无需洗涤细胞、无需溶解产物

• 安全无需刺激性有机溶剂

• 灵活可随时观察显色反应

• 非放射性无需放射性同位素的资格及相应费用


Comparison of CellTiter 96® One Solution and 3H-Thymidine Incorporation Assays

产品规格

• G3582 200 assays = ￥549; ￥2.75 /well

• G3580 1000 assays = ￥1971; ￥1.97 /well

• G3581 5000 assays = ￥6597; ￥1.32 /well

3H -thym id ine cost/96 assay20m l v ial $59 .307m l vial $40 .53P ackard T opcount $31 .44C ellT iter 96 ® G 4000 (1000) $14 .40C ellT iter 96 ® G 4100 (5000) $8 .64C ellT iter 96 ® A Q G 5421 (1000) $12 .96C ellT iter 96 ® A Q G 5430 (5000) $7 .78C ellT iter 96 ® A Q G 5440 (50 ,000) $7 .00M T S P ow der G 1111 (25 ,000) $1 .82C ellT iter 96 ® A QO ne So lution

G 3580 (1000) $16 .00

C ellT iter 96 ® A QO ne So lution

G 3581 (5000) $10 .36

Cost Comparison Cost Comparison CellTiterCellTiter 9696®® Systems to Systems to Radioactive AssaysRadioactive Assays


• 经典的方法

• 非放射性安全节约经费

• 操作简便（G4000 → G5421→ G3581）

• 仅需使用酶标仪

• 价格便宜

活细胞检测

基于琥珀酸脱氢酶的检测基于琥珀酸脱氢酶的检测基于琥珀酸脱氢酶的检测

基于其他氧化还原酶的检测

基于基于基于ATPATPATP的检测的检测的检测

概述

• 均相检测

• 基于氧化还原反应刃天青试卤灵（荧光）

• 仪器：荧光分光光度计、酶标仪

• 适用范围：少量样品高通量筛选

基于其它氧化还原酶的检测

• CellTiter-Blue® Cell Viability Assay G8080 G8081 G8082

荧光刃天青试卤灵试卤灵

氧化还原酶

试剂盒组分: CellTiter-Blue® Reagent

CellTiter Blue Assay Mechanism

Viable Cell

Resazurin ResorufinEmits fluorescence at 590nm

Reduction Rxns

操作步骤

Incubate ~4 hours

CellTiter BlueReagent

Add Reagent to Cells in Culture

Incubate 37°C1-4 hours

Record Fluorescence560/590nm

产品规格

• G8080 20ml = ￥75610 X 96 well plates ≈ 1000 assays ￥0.76/well10 X 384 well plates = 3840 assays ￥0.20/well

• G8081 100ml = ￥215150 X 96 well plates ≈ 5000 assays ￥0.43/well50 X 384 well plates = 19,200 assays ￥0.11/well

• G8082 10 x 100ml = ￥18162500 x 96 well plates ≈ 50,000 assays ￥0.36/well500 x 384 well plates= 192,000 assays ￥0.09/well

Resazurin 的纯度

• 使用高纯度的resazurin（刃天青）

• 混有resorufin（试卤灵）的检测试剂将会增加

实验本底

HPLC Estimates of Resazurin Purity from Two Vendors

Area % = 97.8 & 2.2 Area % = 92.5 & 7.5

• 灵敏度高化学荧光

• 操作简便一步法均相检测加样 - 混合 - 读数

• 非放射性安全节约经费

• 省时

• 便宜

• 需要使用特定仪器荧光分光光度计(酶标仪)

基于其它氧化还原酶的检测

CellTiter-BlueTM Assay 与 MTS assay 基本相似

• Resazurin 和 tetrazolium (i.e. MTT, MTS, etc)均为氧化还原指示剂

• Resazurin assays 的灵敏度稍好– Fig. 5 in TB #317 shows ~400 cell detection for

resazurin in 96 well format at 4 hours, and ~50 cell detection after extended incubation.

• Resazurin assays 可用荧光分光光度计和酶标

仪检测

叠加反应Multiplex CellTiter-Blue and Apo-ONE Assays

• Set up assay plate with test compounds

• Add resazurin

• Incubate 37°C

• Add caspase substrate

• Incubate ambient

• Record fluorescence

CellTiter-BlueTM

Reagent

Apo-ONETM

Reagent

Incubate

Incubate

Record Fluorescence590nm and 530nm

CellTiter-BlueTM can Multiplex with an Apoptosis Assay in the Same Well

活细胞检测

基于琥珀酸脱氢酶的检测基于琥珀酸脱氢酶的检测基于琥珀酸脱氢酶的检测

基于其他氧化还原酶的检测基于其他氧化还原酶的检测基于其他氧化还原酶的检测

基于ATP的检测

基于ATP的检测

• CellTiter-Glo® Luminescent Cell Viability Assay G7570 G7571 G7572 G7573

萤光素 + ATP + O2

萤火虫萤光素酶

Mg2+氧化萤光素 + AMP + PPi + CO2

光

试剂盒组分: CellTiter-Glo® Buffer

CellTiter-Glo® Substrate

CellTiter-Glo® Luminescent Cell Viability Assay

• 均相检测 (加样－混合－读数)

• 检测ATP

• 基于萤光素酶反应

• 第一个均相的ATP检测

• 辉光型信号（半衰期>5小时）

比 CellTiter 96® AQeuous One Solution (MTS) Assay ：

• 更灵敏– CellTiter-Glo ® Assay ≤ 50 cells– CellTiter 96® One Solution ~ 1000 cells

• 更快

- 无需孵育4小时

Sensitive is beautiful, man.

产品规格

• G7570 100 assays =￥ 639; ￥6.39 /well• G7571 10 X 100 assays =￥2961; ￥2.96/well• G7572 1,000 assays =￥2511; ￥2.51/well• G7573 10 X 1000 assays =￥19431; ￥1.94/well

CellTiter-Glo BufferCellTiter-GloSubstrate

“Reagent”

操作步骤

Linear Response at Low Cell Number

Sensitivity !!

CCD Camera Images of ATP Standards in 96 & 384 Format

10µM

1µM

0.1µM

0.01µM

0.01µM

特点

• 灵敏 50个细胞生物荧光

• 简便一步法均相检测加样-混合-读数

• 快速 10分钟

• 灵活 96 or 384, CCD• 安全非放射性节约经费

• 需要使用特定仪器发光检测仪

BacTiter-Glo™ Microbial Cell Viability Assay

-均相的检测方法 ATP

-与 CellTiter-Glo相似，为检测微生物而特别设计

Cells ATP Luminesceneextraction detection

BacTiter-GloTM 试剂作用于不同的微生物有机体

革兰氏阴性革兰氏阳性其它

大肠杆菌金黄色葡萄球菌酿酒酵母

铜绿假单孢菌粪链球菌白色念珠菌

阴沟肠杆菌肺炎链球菌

海床黄杆菌枯草芽孢杆菌

流感噬血菌蜡状芽孢杆菌

普通变形菌藤黄节杆菌

鼠伤寒沙门氏菌

小肠结肠炎耶尔森氏菌

红色：药物开发

绿色：生物防御

紫色：模型微生物

蜃楼耶尔森氏菌白色：其它

Extended range

Sensitivity !!

BacTiter-GloTM: 延伸的检测范围

Antimicrobial activity of oxacillin on S. aureus

0%10%20%30%40%50%60%70%80%90%

100%

0 0.5 1 1.5 2

Oxacillin (ug/ml)

% R

LU v

s no

dru

g co

ntro

l

Oxa (ug/ml) 32 16 8 4 2 1 0.5 0.25 0.12 0.06 0.03 0

MIC

BacTiter-Glo™ 微生物拮抗剂敏感性检测

死细胞检测

• CytoTox 96® Non-Radioactive Cytotoxicity Assay

G1780

• CytoTox-ONETM Homogeneous Membrane Integrity Assay

G7890 G7891 G7892

膜完整性检测

A. 染料排斥法– Trypan Blue– Propidium Iodide / Ethidium Homodimer

B. 细胞内容物的漏出– Lactate Dehydrogenase (LDH) Release– Glucose-6-Phosphate Dehydrogenase– 51Cr-release

细胞毒性检测 – 经典方法

（四唑盐）甲臢甲臢

乳酸脱氢酶 LDH

CytoTox 96® Non-Radioactive Cytotoxicity Assay G1780

NAD+ + 乳酸丙酮酸 + NADH

NADH + INT NAD+ +心肌黄酶

• 经典的方法

• 非放射性安全

• 仅需使用酶标仪比色

• 操作简便

Lactate PyruvateLDH

DiaphoraseResorufin Resazurin

Leaky cell

LDH

NAD+ NADH

CytoTox-ONE™ Assay Overview

• Prepare “Reagent” by adding Assay Buffer to Substrate Mix

• Equilibrate assay plate to 22°C• Add Reagent directly to Assay wells

(1:1 vol)• Incubate 10 min• Add Stop Solution• Mix• Record Fluorescence (560Ex/590Em)

操作步骤

概述

• 经典方法

• 均相检测

• 化学荧光信号

• 节约时间

• 可叠加其它检测试剂

叠加反应 CytoTox 96® & CellTiter 96®

产品规格－CytoTox ONE

• G7890 (2 plate kit) = ￥909 2 X 96 well plates ≈ 200 assays ￥4.54/well2 X 384 well plates = 768 assays ￥1.18/well

• G7891 (10 plate kit) = ￥281710 X 96 well plates ≈ 1000 assays ￥2.82/well10 X 384 well plates = 3840 assays ￥0.73/well

• G7892 (10 plate kit) = ￥281710 X 96 well plates ≈ 1000 assays ￥2.82/well 10 X 384 well plates = 3840 assays ￥0.73/well

经典方法 vs 一步法

经典方法CytoTox 96 Non-Radoactive

一步法CytoTox-ONE

• 转移培养上清 2块板

• 比色法

• 30min 孵育

• 在原来的培养板上直接检测

• 化学荧光信号

• 10min 孵育

实验结果取决于：

• 细胞死亡进程

• 毒素的浓度

• 细胞与毒素作用的时间

• ……

改善细胞毒性实验结果的做法：

• 待检化合物的多个浓度

• 多个细胞-化合物的作用时间

• 复合多个指标 (cytotoxicity, apoptosis)

同时检测活细胞和死细胞

MultiTox-Flour Multiplex Cytotoxicity Assay

MutiTox-Fluor Multiplex Cytotoxicity Assay

Live Cell Protease Assay What exactly is the live/dead assay?

Cytoplasm“Live Cell” Protease

Gly-Phe-AFC

(Ala-Ala-Phe)-R110

Nucleus

Fluorescence

X

Dead Cell Protease Substratedoesn’t appreciably add to live cell signal due to poor permeability

“Dead Cell”Protease

What exactly is the Live-Dead assay?Dead Cell Protease Assay Concept

Nucleus

Cytoplasm“Live Cell” Protease

Gly-Phe-AFC

(Ala-Ala-Phe)2-R110

“Dead Cell”Protease

No cleavagedue to inappropriateenzymatic environment or enzyme lability

Fluorescence or Luminescence

X Insult

What solution do we provide?Simple multiplexed CV/CT assay

Continue with compatible endpoint assays (Apo-ONE, Caspase-Glo, or CellTiter-Glo Assays, total cell number with dye stain

ADDLive Cell &Dead Cell Reagent

INCUBATE15-30 min

READFluorescenceLuminescence

Live Cell Assay (Protease Retention) Correlates with Viability by ATP

A non-destructive v. lytic endpoint

The Ratiometric Response for Data Normalization

An inverse relationshipexists between the live dead cell signals.

Raw or signal to noise

Percentage of maximal signal

What are the key pieces of data?Multiplexed Viability and Total Cell Assay

Viability/Cytotoxicity and Caspase Induction

Cell Lines Examined with the Multiplexed Cytotoxicity Assay

Cell Line Sex Age Histology Source/Origin

HCT M >18 carcinoma colonHL-60 F 36 promyleocytic PBL leukemiaSK-MEL-28 M 51 melanoma melanomaMCF-7 F 69 adenocarcinoma mammaryPA-1 F 12 teratocarcinoma ovaryACHN M 22 carcinoma kidneyPC-3 M 62 adenocarcinoma prostateDU-145 M 69 carcinoma prostateNCI-H226 M na squamous lungLN-18 M 65 glioblastoma brainHeLa F na carcinoma cervixJurkat M na T-cell leukemia lymphocyteHek293 na <1 transformed kidneyHepG2 M 15 hepatocarcinoma liverNK-92CI M 50 lymphoma NK cellU937 M 37 histocystic lymphoma monocyte

如何选择合适的细胞活力检测试剂盒？

• first “What do I really want to measure?”Number of living cells (Viability)

How many live cells are leftNumber of dead cells (Cytotoxicity)

How many cells were killedIncrease in cell number (Proliferation)

• Then ask… “Do I want to determine the mechanism of cell death?”

NecrosisApoptosis

细胞凋亡检测

基于TUNEL的检测

基于Caspace家族成员的检测

复合方法

凋亡相关抗体

凋亡

• 程序化死亡: 在不破坏器官完整性的前提下，去除某些细

胞的协同激活机制。

• 是多数器官保持自稳及正确发育的重要途径

• 异常的凋亡导致病变：癌症神经退行性疾病

• 非炎性反应

• 是一系列蛋白溶解事件的级联反应

凋亡 vs. 坏死

• Membrane blebbing, nuclear and cytoplasmic shrinkage, chromatin condensation.

• Fragment into membrane-bound apoptotic bodies.

• Phagocytosed by macrophages without producing immune response.

• DNA degrades into 180-200 bpfragments (laddering).

• Intrinsic active genetic program.

• Cell swelling, loss of membrane integrity, chromatin flocculation.

• Cell lysis with local inflammatory response.

• DNA degrades into smear.

• Does not require protein synthesis.

Time Zero 30min-1hr 4-6hr 24hr

Viable cell ← apoptosis → 2° necrosis

Viable cell ← necrosis → cell debris

细胞毒性的不同通路

凋亡检测方法

• 形态学观察

• DNA断裂 - TUNEL

• 胞膜改变 - CytoTox-ONE

• 抗体标记物 - Anti-ACTIVE® Caspase-3 pAbAnti-Cytochrome c mAbAnti-PRAP p85 Fragment pAb

• 细胞活力检测 - CellTiter AQ, -Blue, -Glo

• Caspase活性 - Apo-ONE, Caspace-Glo

信息

复杂程度

速度

细胞凋亡检测


基于基于基于CaspaceCaspaceCaspace家族成员的检测家族成员的检测家族成员的检测

复合方法复合方法复合方法

凋亡相关抗体凋亡相关抗体凋亡相关抗体

原理

• 凋亡细胞的DNA出现断裂 180–200bp

• TdT（末端脱氧核糖核苷转移酶）把带有标记物的脱氧核糖核苷加在DNA碎片的3’-OH末端

• 标记物的显色、发光

• DeadEndTM Colorimetric TUNEL System G7360 G7130

• DeadEndTM Fluorometric TUNEL System G3250


TUNELTdT-mediated dUTP Nick-End Labeling

DeadEndTM Colorimetric TUNEL System操作步骤

Attach sections or cells to slide

Fix

Permeabilize

Pre-equilibrate

Label fragmented DNA (biotinylated dNTPs)

Stop reaction

Block endogenous peroxidase

Add Streptavidin HRP

Add DAB

Analyze under light microscope

DeadEndTM Colorimetric Apoptosis Detection System

• 检测DNA 降解 –重要的凋亡信号

• 在细胞混合群体中检测凋亡细胞

• 单细胞水平

• 原位检测

• 非放射性

• 可用于组织切片、培养细胞

DeadEndTM Colorimetric Apoptosis Detection in HL-60 cells

Anisomycin-treated DMSO-treated controls

DeadEndTM Colorimetric Apoptosis Detection in rat brain tissue sections

Staining in the LGN after axotomy-induced cell death

Staining in the contralateralcontrol LGN

DeadEndTM Fluorometric TUNEL System 操作步骤

Fix

Wash

Permeabilize

Pre-equilibrate

Label fragmented DNA (F-dUTP)

Stop reaction

Analyze under fluorescence microscope

Attach cells to slide

Wash

Wash

Stain (PI)

Wash

Fluorescein Apoptosis Detection of HL-60 cells

Anisomycin-treated DMSO-treated controls

DeadEndTM Fluorometric TUNEL System

• Ideally suited for tissue sections

• Morphology can be assessed simultaneously

• Longer assay

• ￥124.4/assay, ￥88.4/assay

• Extremely rapid• Ideally suited for cells

in culture/flow cytometry

• Thick tissue sections have problem with autofluorescence

• Cannot observe morphology

• ￥59.6/assay

TUNEL Assays----Fluorescent vs. Colorimetric

细胞凋亡检测

基于基于基于TUNELTUNELTUNEL的检测的检测的检测




Apoptosis Pathways

MitActive caspase-8

Pro-caspase-8

FADD

Fas/TNF

Receptor Chemicals

Cyto C

Cytosolic factors(Bid)

+Pro-caspase-9 + Apaf-1

Active caspase-9

Caspase-3 and 7

DFF, ICAD, PARP

Staurosporine Etoposide

DNA cleavage and morphological changes

DNA damage?

Z-VAD-FMK

Modified from Sun et al. (1999)J. Biol. Chem. 274: 5053.

Z-VAD-FMK

哺乳动物细胞主要的凋亡途径

Receptor Mediated (Extrinsic)

Mitochondrial(Intrinsic)

Apoptosis Inducing Factor

Procaspase-8 Procaspase-9 Non-caspase (?)

Caspase-3 and-7

Caspase-9Caspase-8Digestion of Cell Scaffold Proteins, DNA laddering and finally, DEATH“Death Effectors”

“Apical or Initiator”Caspases

Committed cellular fate

原理

• 哺乳动物细胞凋亡时caspace酶的活性增加

• 将caspace酶的特异底物修饰（发色基团、萤光前体）或在酶反应中引入ATP

• 显色、发光


• CaspACETM Assay System, Colorimetric G7351 G7220比色法


化学荧光法• CaspACETM Assay System, Fluorometric G3540• Apo-ONETM Homogeneous Caspase-3/7 Assay

G7792 G7790 G7791

生物发光法• Caspase-GloTM 3/7 Assay G3540• Caspase-GloTM 8 Assay G8200 G8201 G8202• Caspase-GloTM 9 Assay G8210 G8211 G8212

Apo-ONETM Homogeneous Caspase-3/7 Assay

Z-DEVD-R110-DEVD-Z

Caspase 3/7

Z-DEVD +

R110

One Step, “加样－混合－读数”


配制试剂

加样

混合

读数


Sensitivity !!

Caspase-GloTM Assays

Z-XEXD-

Z-XEXD-

Caspase

Luciferase

2

Light

+ConsensusTetrapeptidesDEVD: Casp-3/7LETD: Casp-8LEHD: Casp-9

改构萤光素

萤光素

Simplicity of Homogeneous Format

Dead CellViable Cell

Caspase-Glo™Reagent

Reagent No RxnX

Reagent No RxnX

Apoptotic Cell

Reagent Luminescence

InactiveCaspase

InactiveCaspase

ActiveCaspase

• Caspase Substrate • Lysis Solution

Caspase-GloTM Assay Protocol

配制试剂

加样、混合

读数

Caspase-GloTM 3/7 Assay: 线性范围和灵敏度

Luminescence is Proportional to Caspase 3 Activity

0.01

0.1

1

10

100

1000

10000

100000

0.0001 0.001 0.01 0.1 1 10 100

Caspase-3 mU/ml

RLU

(bck

gr s

ub)

Titration 1Titration 2

R2=.99Slope=.99

R2=.99Slope=1.1

Caspase-GloTM 3/7 Assay: 稳定性

0.1

1

10

100

1000

10000

100000

0.0001 0.001 0.01 0.1 1 10caspase 3 (mU)/well

RLU

(bac

kgr s

ubtra

ct)

.5 hr read2 hr read4 hr read6 hr read

Caspase-GloTM 3/7 Assay 应用

• 纯酶检测– Caspase inhibitor screens– IC50 analysis of potential inhibitors

• 细胞培养物检测

– Apoptosis assays– Drug screening for apoptosis activation or inhibition– Drug screening for Caspase 3/7 activation or

inhibition– Secondary Cytotoxicity Screening

Caspase-GloTM 3/7 Assay:

• 已测试过的细胞系与诱导剂– 细胞: Jurkats, L929, HeLa, HL-60, SH-SY5Y, HepG2– 诱导剂: anti-Fas, TNFα, staurosporine, clozapine, vinblastine,

tamoxifen网上工具软件：apop. assistant

• 可检测血清中的 caspase-3 活性– We recommend a no cell control– Background can be reduced if cells can be grown in serum-

reduced medium

细胞凋亡检测



复合方法


复合方法

Death CheckTM I Assay System G7370CaspACETM Assay System, Colorimetric G7351 DeadEndTM Colorimetric TUNEL System G7360

Death CheckTM II Assay System G7380CellTiter 96® AQueous One Solution Cell Proliferation Assay G3582DeadEndTM Colorimetric TUNEL System G7360

Death CheckTM III Assay System G7390CaspACETM Assay System, Colorimetric G7351 CellTiter 96® AQueous One Solution Cell Proliferation Assay G3582

Viable Cell

Time Zero 30min-4hr 24hr

Viable Cell

Caspase 0 0 0LDH Release 0 +++ ++ATP +++ 0 0MTS +++ 0 0Resazurin +++ 0 0

Apoptosis

Necrosis NecroticDebris

Apoptotic

Caspase 0 +++ ++LDH Release 0 + +++ATP +++ ++ 0MTS +++ ++ 0Resazurin +++ ++ 0

Apoptotic bodies& Debris

细胞凋亡检测




凋亡相关抗体

凋亡相关抗体

• Anti-ACTIVE® Caspase-3 pAb G7481

• Anti-Cytochrome c mAb G7421

• Anti-PRAP p85 Fragment pAb G7341

• 免疫组化

• 免疫细胞化学

• Western blotting

• 流式细胞分析

Anti-ACTIVE® Caspase-3 pAb staining of anti-Fas induced Jurkat cells (human)

Control untreated Apoptotic

Anti-ACTIVE® Caspase-3 pAb staining of TNF alpha treated L929 fibroblasts (mouse)

Control cells 5.5hr TNF alpha 5.5 hr

Anti-ACTIVE® Caspase-3 pAb staining of staurosporine treated SH-SY5Y cells (human neuroblastoma)

Control, 5hr 0.5uM stauro., 5hr

信号转导相关的检测

磷酸激酶检测

磷酸酶检测

• 细胞对外部刺激的反应

• 信号 = 细胞间传递信息的生化反应

• 细胞反应性的改变 = 信息从胞膜—胞浆– 胞核

– 基因转录

• 涉及蛋白的修饰作用

激酶的磷酸化作用

磷酸酶的去磷酸作用

蛋白酶的切割作用

信号转导

Plasma membrane

or

or

+

PKAAKAP

PLCβ PLCγ

CaNCaMKII

CaMKK

CaMCaMKIV

PI3KAkt

CREB

PKCs

survival

transcription

PP2A

PTEN

MAPKs

MEKs

MEKKs

SosGrb

Shc

Organelle/cytoskeleton

nucleus

ELK-1

ATF-2c-JUN

AP-2

IkBNFκB

NFκB

SP1

真核细胞信号网络

IKK

14-3-3Bad

+

cAMP

Ca2+

Ca2+

Ca2+

Ca2+

GPCRsDAG IP3 R

TKs

AC I-IX

Gβγ

Gsα

Gqα

Go/iα

Ras

PIPs

Gβγ

Rap1Epac

PPI

磷酸激酶检测相关产品

磷酸激酶检测相关产品

• 酶

• 底物

• 抑制物

• 抗体

• 酶活性检测试剂盒

KinaseKinase--GloGlo®® Luminescent Luminescent KinaseKinase AssaysAssays

SignaTECT® Assay Systems

PepTag® Non-Radioactive Assays

萤光素 + ATP + O2

萤火虫荧光素酶

Mg2+氧化萤光素 + AMP + PPi + CO2 +

光

原理

激酶反应

Kinase-GloTM Luminescent Kinase Assay

Kinase-GloTM Luminescent KinaseAssay

• Kinase-GloTM Buffer

• Kinase-GloTM Substrate

试剂盒组分:

• Kinase-Glo™ 检测激酶反应中产生的ATP来反映激酶活性

• 含量与激酶活性成反比

• 很好的 Z` 值

Kinase-Glo™ Product Description

操作步骤

• 激酶反应– Kinase, substrate, +/- compounds– Start reaction with ATP– Mix and incubate at RT

• 加入等体积 Kinase-Glo™ Reagent• 混合（15 sec）• 孵育（10 min）• 读取发光强度

操作步骤

特点

• 均相检测加样 - 混合 - 读数

• 简便、灵敏、非放射性

• 底物可以是多肽、蛋白、脂类

• 不需要标记反应组分

• 生物发光信号强

• 动力学范围广、Z’值高

• 精确的IC50值

Promega’s cell based assay products

Cell Viability / Cytotoxicity Assays

• CellTiter-Glo® Assay• CytoTox-ONE™ Assay • CellTiter-Blue® Assay• CellTiter 96® AQueous One

Solution • Live Dead Assay

Apoptosis Assays• Caspase-Glo 3/7® &

Caspase-Glo® 8 & 9 Assays• Apo-One® Caspase-3/7

Assay

Reporter Gene Assays• Dual-Glo® Luciferase Assay• Dual-Luciferase® Reporter

Assay• Chroma-Glo™ Luciferase

Assay• Bright-Glo™ and Steady-

Glo® Luciferase Assays• pGL4 Vector series• uHTS Dual Reagent

GloMax™ Luminometers

谢谢！谢谢谢！谢！

驶入细胞分析的高速路

Documents

Transcript of 驶入细胞分析的高速路