[Cytometry] effect of mitogen

Roberts, Daniel Daniel.Roberts3 at bms.com
Wed May 4 13:52:45 EDT 2011


Snehal,

Scatter properties of mitogen stimulated lymphocytes gets quite crazy, as they a-synchronously proliferate (unless controlled), and some cells become apoptotic due to anergy (Example PMID's: 8547710, 16080188; Also, http://cancerres.aacrjournals.org/content/37/12/4635). This explains why you lose the classic scatter profile after stimulating the cultures.

In general it would be good to know if you're working with whole blood cultures, or are separating out and stimulating only mono-nucleated cells. Also, what is the purpose of this study? Do you want to simply evaluate effects on cell cycle? If so a 72-hour exposure may not be appropriate, even at ng dose levels.

The media will depend upon what your cells like. RPMI works for most things, but I've found that Human lymphocytes are quite happy in Williams E Medium. It all depends on your experimental conditions, and it would be wise to try different concentrations of serum.

CD45 will only help if you need it to pull leukocytes away from RBCs during acquisition. However, this depends if you're a) fixing your cells, and more importantly on b) what you're fixative is. If using alcohol, you'll likely denature/destroy you're CD45 epitope. The second Pubmed paper referenced above describes a protocol for preserving the extracellular epitopes when staining intracellularly, if this is your goal.

For your nucleic acids stain, PI is great for cell cycle, but use in the presence of RNAse (DNAse free), in cells that are permeabilized. 7-aad, draq 5, ruby dye cycle, or any other cell permanent DNA-specific stain would be fine as long as it fits in your panel. The fourth edition of Howard Shapiro's Practical Flow Cytometry has a lot of information regarding different nucleic acid dyes. Also there is some info that may help you online through Invitrogens/Molecular Probes Handbook.

Dan

______________________________
Daniel J Roberts MS, Research Scientist
Bristol-Myers Squibb, 1 Squibb Drive
83-1-21, New Brunswick NJ 08901
Reply to: Daniel.Roberts3 at bms.com

>-----Original Message-----
>From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-
>bounces at lists.purdue.edu] On Behalf Of snehal mhatre
>Sent: Monday, May 02, 2011 11:53 AM
>To: cytometry at lists.purdue.edu
>Subject: [Cytometry] effect of mitogen
>
>We want to study effect of different concentrations of mitomycin C (MMC) on
>lymphocytes. so for this purpose, we separate PBMCs, and culture them for
>72hrs with different concentrations of MMC. and then check viability of
>cells with PI. for baseline, we check viability of cells on day 0. but after
>72hrs, might be due to culturing of cells, the FSC vs SSC scatter is not
>proper. so it is difficult to gate lymphs and hence checking viability is
>not specific, as we are not sure whether we are gating specific population.
>
>so my queries are
>
>1) for culturing we just adjust cell count with complete media (RPMI-1640)
>and add MMC. Do we have to take any specific precautions during this
>procedure, to maintain the cell morphology
>2) Will adding a marker like CD45 will be of help?
>3) Should we use 7-AAD instead of PI?
>
>Thanks
>--
>Regards
>Snehal Mhatre
>PhD student
>NIIH, Parel
>Mumbai
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