Guide to Cell Proliferation and Apoptosis Methods

互联网2013-09-05

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Chapter 1: Cell Death - Apoptosis and Necrosis

1.1	Introduction	2
1.1.1	Terminology of cell death	2
1.1.2	Differences between necrosis and apoptosis	3
1.1.3	Apoptotic Pathways	4

1.2	Apoptosis Assay Methods	6
	Method/Product selection guide	7
1.2.1	Methods for studying apoptosis in cell populations	8
1.2.1.1	Assays that measure DNA fragmentation	8
	Apoptotic DNA Ladder Kit	11
	Cell Death Detection ELISA^PLUS	13
1.2.1.2	Assays that measure apoptosis-induced proteases (caspase)	16
	Caspase 3 Activity Assay	17
	Anti-PARP	20
1.2.1.3	Summary of methods for studying apoptosis in cell populations	22
1.2.2	Methods for studying apoptosis in individual cells	24
1.2.2.1	The TUNEL enzymatic labeling assay	24
	In Situ Cell Death Detection Kit, Fluorescein	27
	In Situ Cell Death Detection Kit, AP	29
	In Situ Cell Death Detection Kit, POD	29
1.2.2.2	Assays that measure membrane alterations	31
	Annexin-V-FLUOS	32
	Annexin-V-FLUOS Staining Kit	32
	Annexin-V-Alexa^TM 568	32
	Annexin-V-Biotin	34
1.2.2.3	Assays that use DNA stains	36
	DAPI, Ethidium bromide, Propidium iodide	36
1.2.2.4	Summary of methods for studying apoptosis in individual cells	38
1.2.3	Detection of apoptosis-related proteins	40
	Anti-Fas (CD95/Apo-1)	41
	Anti-Fas-Biotin (CD95/Apo-1)	42
	Anti-Bcl-2 oncoprotein, human	44
	Anti-p53 Protein, mutant	46
	Anti-p53 Protein pan	46
	Anti-p53 pan	46
	Anti-p53, Biotin labeled	46
	Anti-p53, Peroxidase labeled	46
	p53pan ELISA	48

1.3	Cytotoxicity Assay Methods	50
1.3.1	Relationship between cytotoxicity, apoptosis, and necrosis	50
1.3.2	Methods for studying cytotoxicity	50
1.3.2.1	Assays the measure plasma membrane leakage	51
	Cytotoxicity Detection Kit (LDH)	52
	Cellular DNA Fragmentation ELISA	54
1.3.2.2	Assays that measure metabolic activity	58
	Cell Proliferation Kit I (MTT)	58
	Cell Proliferation Kit II (XTT)	58
	Cell Proliferation Reagent WST-1	58
1.3.2.3	Summary of methods for studying cytotoxicity	60

Chapter 2: Cell Proliferation and Viability

2.1	Introduction	64
2.1.1	Terminology of cell proliferation and viability	64
2.1.2	Cell cycle	64

2.2	Cell proliferation/viability assay methods	67
	Method/Product selection guide	68
2.2.1	Methods for studying cell proliferation and viability in cell populations	70
2.2.1.1	Assays that measure metabolic activity	70
	Cell Proliferation Kit I (MTT)	73
	Cell Proliferation Kit II (XTT)	74
	Cell Proliferation Reagent WST-1	75
2.2.1.2	Assays that measure DNA synthesis	77
	BrdU Labeling and Detection Kit III	79
	Cell Proliferation ELISA, BrdU (colorimetric)	81
	Cell Proliferation ELISA, BrdU (chemiluminescence)	81
2.2.1.3	Summary of methods for studying cell proliferation and cell viability in cell populations	84
2.2.1.4	Single reagents for the measurement of DNA synthesis	84
2.2.2	Methods for studying cell proliferation and viability in individual cells	86
2.2.2.1	Assays that measure DNA synthesis	86
	BrdU Labeling and Detection Kit I	87
	BrdU Labeling and Detection Kit II	87
	BrdU Labeling and Detection Kit, FLUOS	88
	In situ Cell Proliferation Kit, FLUOS	90
	In Situ Cell Proliferation Kit, AP	93
	Anti-BrdU, formalin grade	93
	Anti-BrdU-Fluorescein	93
	Anti-BrdU-AP, F(ab)2 fragments	93
	Anti-BrdU-Peroxidase, Fab fragment	93
2.2.2.2	Assays that monitor expression of cell cycle-associated antigens	96
	Monoclonal antibodies to cell cycle-associated antigens	97
2.2.2.3	Summary of methods for studying cell proliferation and viability in individual cells	100

Chapter 3: Appendix

3.1	Technical tips	104
3.1.1	Selected frequently asked questions (FAQs) about cell death assays	104
3.1.2	Technical tips on the TUNEL method	105
3.1.2.1	TUNEL: Improvement and evaluation of the method for in situ apoptotic cell identification	105
3.1.2.2	TUNEL protocol for tissues which tend to give false positives	105
3.1.2.3	Tips for avoiding or eliminating potential TUNEL labeling artifacts	107
3.1.3	Technical tips on the use of Annexin-V-Biotin for light microscope detection	109
3.1.4	Technical tips on the use of the Apoptotic DNA Ladder Kit on tissue samples	109
3.1.5	Technical tips on the Cell Proliferation ELISA kits	110

3.2	Special applications of cell death and cell proliferation methods	111
3.2.1	TUNEL assays	111
3.2.1.1	Discrimination between dead and viable apoptotic cells using two-color TdT assay and surface labeling as detected by flow cytometry	111
3.2.1.2	The use of flow cytometry for concomitant detection of apoptosis and cell cycle analysis	111
3.2.1.3	Comparison of two cell death detection methods: In situ nick translation and TUNEL	112
3.2.1.4	Fixation of tissue sections for TUNEL combined with staining for thymic epithelial cell marker	112
3.2.2	Metabolic assays	113
3.2.2.1	Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts	113
3.2.3	Annexin assays	113
3.2.3.1	The use of annexin for concomitant detection of apoptosis and cellular phenotype	113
3.2.4	BrdU assays	114
3.2.4.1	Detection of bromodeoxyuridine in paraffin-embedded tissue sections using microwave antigen retrieval is dependent on the mode of tissue fixation	114

3.3	References	115
3.3.1	Apotosis- related parameters - Abbreviations and References	115
3.3.2	Examples for applications for Boehringer Mannheim products	120
3.3.3	General references	127