PROPIDIUM IODIDE CELL CYCLE ASSAY

Tim Kute tkute at wfubmc.edu
Fri Jun 22 09:02:39 EDT 2007


When we fix cells with 70% cold ethanol, we first centrifuge the cells to a pellet and
bring the pellet up in 100ul of PBS. Then I add the DNA stain ( PI, buffer, RNAase, NaCl)
to the cells while mixing using a vortex.  This prevents clumps and usually results in a
good stained population.  


Tim Kute 

-----Original Message-----
From: Sylvain GIMMIG [mailto:cytometry at gimmig.org] 
Sent: Wednesday, June 20, 2007 8:57 AM
To: cyto-inbox
Subject: PROPIDIUM IODIDE CELL CYCLE ASSAY

Dear colleagues,

We have two questions:

1. We have been following a standard protocol for the PI cell cycle assay  following
stimulation of human CD4 cells or Jurkat cells with CD3:CD28 antibody-coated Dynal beads
(Pan mouse IgG Cellcept beads). However, the cells seems to be very fragile following
fixation in 70% ethanol (either at 4C or -20C and for periods varying from  either 1 hr
to days of fixation). We are losing many due to lysis.	Does someone have suggestions on
how to reduce cell loss due to lysis at this step?

2. What are the best time points to measure in analyzing the cell cycle in human
CD4 and CD8 cells following stimulation with CD3:CD28 antibodies?

Thanks for any input,


--
Sylvain GIMMIG
Flow Facility Manager
HÔPITAL ST-LUC/CHUM
Bureau:409
264, Boul René-Lévesque est
Montréal, QC, H2X 1P1, Canada
Téléphone: 514-890-8000 ext 35778
FAX: 514-412-7314
Website:http://www.chumtl.qc.ca




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