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peripheral mononuclear cells handling

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peripheral mononuclear cells handling

Postby claire84 » Thu Nov 01, 2012 12:18 pm

Hi guys,
maybe it's a silly question, but I'm not a cellular biologist and I need some help.
I isolated PBMC from the whole blood and then I seeded them in a 96 well plate. After incubation of 24h I would like to monitor the presence of a certain protein on their surface using a labelled antibody.

Do I need to treat them as adherent cells using trypsin before adding the mix with the antibody or can I resuspend them pipetting?
What do yo think?

thanks a lot!
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Postby biohazard » Fri Nov 02, 2012 8:46 am

Depends on what subset of PBMCs you want to study. In general, blood cells are suspension cells and do not require trypsination, but monocytes are an exception: they adhere to certain types of plastics (which includes most cell culture plates) and must be removed with PBS/EDTA & cell scraper, or perhaps with trypsin.

For other PBMCs resuspending them with a pipette is sufficient. The tendency of monocytes to adhere to culture plates can also be easily used to reduce the contamination by monocytes if you want to study B or T cells only.
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Re: peripheral mononuclear cells handling

Postby claire84 » Fri Nov 02, 2012 2:56 pm

Indeed want to study monocytes. I think I'm going to use PBS/edta and to scrap them with the tips of the pipette.
Do you think it's a good idea?
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Postby biohazard » Mon Nov 05, 2012 1:11 pm

Scraping them with a pipette might be bit difficult, but if you have to use 96-well plates then that might be the only option. Or do they have mini-scrapers for so small wells? You could try seeding the cells on a bigger plate and using plastic cell scrapers after the EDTA-treatment. I have used 5 mM EDTA in PBS (pH 7.2) and incubated my cells for some 5 to 15 mins in +4C with moderate success (some of the cells tend to die, probably because of the scraper).

The monocytes require an hour or two in +37C in culture medium to attach to the plate surface, and thus after 24h they should be firmly stuck and you can wash the other cell types away so that they do not interfere with your monocyte analysis.
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