Three-Color Analysis of DNA
LAADAMS@am.pnu.com
Wed, 23 Oct 1996 14:58:00 -0400
Using flow cytometry, I am attempting to perform concomitant analysis of
cell cycle, DNA synthesis and apoptosis in HT29 cells, a human colon
adenocarcinoma cell line which grows in adherent monolayers. Currently, I
am working out protocols for each step individually: 1) monitoring
apoptosis using the TUNEL reaction in Boehringer Mannheim's In Situ Cell
Death Kit-FITC; 2) monitoring DNA synthesis by BrdU incorporation followed
by a-BrdU (Becton Dickinson) and a secondary a-mouse-PE (Jackson); 3)
determining cell cycle positions by 7-AAD incorporation. Briefly, I
synchronize cells using a mitotic shake and overnight treatment with
hydroxyurea. The following morning I wash out the HU, allow cells to
progress through S phase, then treat with a potential anti-cancer agent
once all cells are in G2/M. I am having a couple of technical difficulties
to which I haven't found answers in the literature. First, does anyone
know of a fixation protocol which will work in HT29 cells (or another
adherent mammalian cell line) which will allow me to look at DNA synthesis,
apoptosis and cell cycle all in one culture of cells? Second, I get a
large amount of nonspecific binding of the a-BrdU antibody in cells treated
with HU. Any suggestions?
Lisa Adams
LAADAMS@am.pnu.com
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