Nate Regimbal and other curious parties, Milena Cankovic did some apoptosis studies here in 1994 using the sub-G1 technique. First of all, murine lymphocytes from spleen and lymph nodes were being studied, which were >97% in the G0/G1 phase. This is I think a key issue when trying to use this protocol, since if you are working with cultured cells, for example, you have a broad spectrum of cycling cells. An apoptotic G2 cell could have the same DNA content as a viable G1 or S cell, making it impossible to measure by this protocol alone. As far as determining apoptotic cells from apoptotic bodies, we decided to only count events with DNA index greater than 0.5. (Confirmation was done by DNA laddering.) The sub-G1 peaks we observed were still gaussian, separated from the G1 peaks, and had higher CVs than the G1 peaks. As far as the "nuts and bolts" go, I think I put G0/G1 in channel 500 on a 1024 linear scale, and triggered on DNA content above channel 50 (DNA index 0.1, a "cutoff" I think Dr. Zbigniew Darzynkiewicz once proposed for determining apoptotic cells). I think there are better methods for detecting apoptosis today. Probably the only reason anyone today considers the sub-G1 method is because it is relatively inexpensive. Eric >Hi all, > >How can one be sure that the sub G1 events are apoptotic events, and not just >DNA fragment clusters of varying sizes? Wouldn't marker statistics of the >sub-G1 'pile' also be inaccurate, as the marked events are not actually cells, >but fragments of DNA? Also, regarding the presence of a sub-G1 'apoptotic' >peak: what would cause apoptotic cells to all display slightly lower, yet >Gaussian, DNA content? Doesn't a sub-G1 peak mean that these cells all lost >the same, small amount of DNA? My apologies if this has been covered in the >past. > >Cheers, >Nate Regimbal >PPD Discovery >Menlo Park, CA > >Tim Kute wrote: > >> In most cases, I like to have my G1 peak start in the 1/3 rd of the x axis >> range. If you are looking for apoptotic cells, you might want it higher >> around 2/3 rd of range. If you really want to see every event, you might >> even want to put the scale on log and have the G1 peak at the 80% area of >> the x axis. >> >> Tim Kute >> >> Maciej Simm wrote: >> >> > I'm posting this on behalf of a collegue. She's doing an assay with >> > just PI looking at the peaks before/after incubation of her cells in >> > pro-apoptotic drugs. If anyone looks at PI on a Calibur, could you >> > fill me in on the instrument settings that work the best? I would >> > appreciate any other tips. >> > >> > Maciej >> > >> > >find out voltage settings for the PI assay. in particular ask about >> > >a printout of an acquisition template they use. also - >> > >> > >fsc lin or log? what voltage? >> > >ssc same as above >> > >fsc s.a.a. >> > >fl2 s.a.a. >> > >fl3 s.a.a. >> > >> > >what about compensation? >> > >> > __________________________________________________ >> > Do You Yahoo!? >> > Make international calls for as low as $.04/minute with Yahoo! Messenger >> > http://phonecard.yahoo.com/ > >______________________________________________________________________ >This email transmission and any documents, files or previous email >messages attached to it may contain information that is confidential or >legally privileged. If you are not the intended recipient or a person >responsible for delivering this transmission to the intended recipient, >you are hereby notified that you must not read this transmission and >that any disclosure, copying, printing, distribution or use of this >transmission is strictly prohibited. If you have received this >transmission in error, please immediately notify the sender by telephone >or return email and delete the original transmission and its attachments >without reading or saving in any manner. >______________________________________________________________________ Eric Van Buren, aa9080@wayne.edu Karmanos Cancer Institute and Immunology & Microbiology Wayne State University, Detroit, Michigan, USA
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