University:
Boston University
Course:
CAS BI 108 | Biology 2
Academic year:

2023
Views:

468

Pages:

1
Author:

Carina Watson

General flow cytometry methods for apoptosis and cell cycle arrest 1. Apoptotic profiles Cells are fixed in 70% ethanol in serum-free RPMI1640 medium overnight, washed in phosphate buffered saline and then stained with propidium iodide in the presence of RNAse. Events are detected by flow cytometry on the FL2 channel with the detector in logarithmic mode (FL2-H). Doublet discrimination on the FL2 channel is used to identify single cell events and 40,000 events are collected at a low flow rate. EXPECTED RESULTS: The appearance of events with sub-G1 DNA content is consistent with the accumulation of apoptotic cells. 2. Cell cycle arrest. Cells are fixed and stained as above. Events are detected by flow cytometry on the FL2 channel with the detector in linear mode (FL2-A). Doublet discrimination on the FL2 channel is used to identify single cell events and 40,000 events are collected at a low flow rate. Cells should be exposed to a DNA damaging drug (adriamycin, a common anti-cancer compound) as a positive control. EXPECTED RESULTS: The data should show the emergence of a G2/M phase cell cycle arrest over time, and a sub-G1 population, consistent with apoptosis. 3. Six color stains. Emission spectra of different fluorochromes are chosen in such a way that spectral overlap is minimized. Bandpass filters optimize the photons collected, while minimizing overlap. Any remaining overlap is typically handled with electronic compensation.