Hi Ulrika, On Fri, 6 Sep 2002, Ulrika K Eriksson wrote: > Concerning cell cycle analysis: > -After fixtion in EtOH the cells should be treated with RNase. Does > the RNase have to be DNase free? I use Ribonuclease A from Sigma (R5125). Since the aim of the game is to remove dsRNA, it is best to have minimal DNase contamination. > -Is it necessary to buy unstained trout red blood cells (TRBC) to use > as a control for the method/flow cytometer or are TRBC only used in > clinical applications? Internal DNA standards are useful in clinical applications or if you expect aneuploid populations in your cultures. For most applications using cell lines, an exponentially growing culture will allow you to position your G1 peak for that cell line. > -Would a certain channel number be recommended for cell cycle > analysis (e.g 255,512,1023)? On a 1024 channel scale, I would place the G1 peak somewhere around channel 200-250. The reason for this is that is there is any reduplication leading to 8n cells, they would still be on scale and not disappear into channel 1023. > Concerning apoptosis studies: > -To set up the flow cytometer and compensate for crosstalk I'm going > to use apoptosis-induced cells stained with FITC only and > apoptosis-induced cells stained with PI only and run them separetly > on the flow cytometer. I was recommended to use butyric acid (5mM) as > an apoptosis inducer. Does anybody know an approximate incubation > time to achieve an "apoptosis" maximum (i.e. where the signal will be > the strongest)? I assume you are using something like annexin-FITC and using PI to exclude dead cells? If so, then I would do as you describe (its more important to have an FITC control and the spillover is greater) before running your dual-stained samples. Not so sure I can help with the second part although using physiological concentrations of butyrate on colonic cancer cell lines, we dont see a peak of apoptosis until 48 hours after incubation. Hope that helps! Derek ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Cancer Research UK, e_mail:derek.davies@cancer.org.uk London Research Institute, mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux *************************************************************************
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