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APOPTOSIS I
[adapted from: MA Hotz, J Gong, F Traganos, and Z Darzynkiewicz.
1994. Flow cytometric detection of apoptosis: Comparison of the
assays of in situ DNA degradation and chromatin changes. Cytom
15:237-44.]
Requires:
HBSS = Hanks balanced salt solution
CAM = camptothecin, DNA topoisomerase I inhibitor, 5 mg/ml in DMSO
(stock) [as positive control]
fix = 1 ml HBSS/9 mls 70% ETOH
SC Buffer = 0.05 M Na2HP04 (9 parts)/25 mM citric acid (1 part)/0.1 %
Triton X-100, pH 7.8
PI Buffer - 10 mM PIPES buffer(Calbiochem)/0.1 N NaCl, 2 mM MgCl2/0.1
% Triton X-100, pH 6.8 with 20 mg PI/50 U-ml-1 RNase A
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1. For positive apoptotic cells: incubate cells with 0.15 mM CAM
for up to 3 h.
2. trypsinize quickly, add cold media, centifuge quickly at 4 C
3. fix (1x10 7 cells/10 ml), 24-48 h, -20 0C
4. pellet, 200 g, 5 min
5. resuspend in 1 ml SC Buffer
6. stain by adding .2 ml SC suspension to 1.5 ml PI Buf, incubate
(at least) 30 min, in dark
Apoptosis II (alternative):
[adapted from: WG Telford, LE King, PE Fraker. 1992. Comparative
evaluation of several DNA binding dyes in the detection of
apoptosis-associated chromatin degradation by flow cytometry. Cytom
13:137-43.]
Requires:
PI - 0.1 % Triton X-100//0.1 mN EDTA(NA)2//50 mg/ml RNase A (50
U/mg)//50 mg/ml PI
1. fix 2x106 cells, chilled to 40C, rapidly resuspending in 80 %
ETOH, 4 0C.
2. incubate on ice 30 min
3. pellet 1200 rpm, 5 min
4. wash 1x PBS, pellet 1200 rpm, 5 min
5. resuspend in PI, in dark, analyze within 24 h
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