Hollier, Mark J. (CDC/CCID/NCZVED) (CTR) etu4 at CDC.GOV
Wed Jun 20 14:12:45 EDT 2007

Hi Sylvain,

I am currently working with human PBMCs, and I have not come across a loss of cells
during permeabilization. The protocol that is being used, are they fixing and
permeabilizing with 70% ethanol only? I personally use 4% paraformaldehyde final
concentration to fix (either use 10min at 37C, 30min at RT, or 45 min on ice), and then
permeabilize with 70% ethanol. This has never given me a problem with cell lysis. For the
permeabilization I keep the cells on ice for 30 min, then either use them immediately or
store them at -20C. I also use Jurkat cells for some controls and assay setup, but again
have never seen lysis with the method described above. The fixation with the
paraformaldehyde does not interfere with cell cycle analysis (as I am doing this right
now), and is also good for extracellular and intracellular staining.

Another thought about the cell loss, are you sure it is due to cell lysis? In the past I
have come across loosing cells during the centrifugation steps, especially when they are
in the ethanol following fixation and permeabilization. They just seem difficult to
pellet in the ethanol. Before fixation I typically use 400g for 10mins, after fixation
and permeabilization I use 1000g for 10mins. This stopped the cell loss. Another
potential source for cell loss is whether the user is transferring the cells from one
tube to another at the various steps in the protocol, this can result is substantial cell
loss. When I perform my experiments I seed the cells in the FACS tubes (12mm x 75mm, snap
cap) and perform all subsequent steps in the same tube, including the analysis.

As for the best time point, the only way to know for your particular samples and set-up
(including operator variabilities) is to perform a time course experiment, assaying
perhaps every 4 or 8 hours for a week. If using primary PBMCs they will be in the G0
stage, and will not move through the cell cycle unless you stimulate them. How much to
stimulate, and which stimulation agent (as most are biased towards particular subsets of
PBMCs) needs to be assessed. One way of getting positive controls for the cell cycle is
to use drugs to stop the cells in a particular phase of the cell cycle, such as
Aphidocolin to stop the cells in G1/S and Nocozadole to stop the cells in G2/M. 

Hope this is of help to you,


Dr Mark Hollier
Centers for Disease Control and Prevention,
Coordinating Center For Infectious Diseases,
National Center For Zoonotic, Vector-Borne, & Enteric Diseases,
Division Of Viral & Rickettsial Diseases,
Chronic Viral Diseases Branch,
Atlanta, GA, 30329
E-mail:etu4 at
Tel: 404-639-2276
Fax: 404-639-3540

-----Original Message-----
From: Sylvain GIMMIG [mailto:cytometry at] 
Sent: June 20, 2007 08:57
To: cyto-inbox

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
264, Boul René-Lévesque est
Montréal, QC, H2X 1P1, Canada
Téléphone: 514-890-8000 ext 35778
FAX: 514-412-7314

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