I'm isolating human PBMCs, incubating them for a week with a fungal pathogen and then hope to run them through flow to see what's going on with the B and T cells, but am not having much luck. Cells are adhering to the bottom of the wells, but could they be macrophages?
Any advice would be immensely helpful. Forgive me if this is too basic. :-/
Hi all,
Both human B and T cells are suspension cells, although they do express several adhesion molecules that they use in tissue homing and chemotaxis. PBMCs also contain monocytes, which in turn adhere on most plastic surfaces. So if you see cells sticking firmly to the cell culture vessel surfaces, they are monocytes.
Naturally, under normal conditions, the B and T cells sediment to the bottom of the cell culture vessel, but gentle agitation or pipetting is enough to bring them back to suspension. Trypsination is definitely not needed for these cells.
Macrophages are not present in venous blood, but monocytes can mature into macrophages under suitable conditions.
As for the lack of luck in your experiments: I am not sure what medium/cytokines/supplements you use in your culture, but B cells generally fare poorly in "standard" PBMC culture conditions when compared to T cells, and therefore could be depleted by day 7.
Hey Tiffany,
I just started to work with PBMCs and what I observe is that the cells are semi-adherent. The attachment to the flask is not very strong and I resuspend them very well before I use them for my FACS experiment. I don't need to use trypsin. Also, I culture them in a T75 flask in upright position.
Hope my answer could help a litte.
Yes (though not like monocytes/macrophages are)- they are also differentially adhesive-there are older methods to enrich B cells this way
Hi all,
Both human B and T cells are suspension cells, although they do express several adhesion molecules that they use in tissue homing and chemotaxis. PBMCs also contain monocytes, which in turn adhere on most plastic surfaces. So if you see cells sticking firmly to the cell culture vessel surfaces, they are monocytes.
Naturally, under normal conditions, the B and T cells sediment to the bottom of the cell culture vessel, but gentle agitation or pipetting is enough to bring them back to suspension. Trypsination is definitely not needed for these cells.
Macrophages are not present in venous blood, but monocytes can mature into macrophages under suitable conditions.
As for the lack of luck in your experiments: I am not sure what medium/cytokines/supplements you use in your culture, but B cells generally fare poorly in "standard" PBMC culture conditions when compared to T cells, and therefore could be depleted by day 7.
Thanks, everybody, for your feedback. Currently we are culturing in 96-well plates with RPMI + 10% human serum and are not adding any supplements throughout the week. After consideration, I think the cells need more surface area and possibly additional nutrients a few days post-infection.
I welcome any other thoughts or tips. Thank you again so much!
How many cells per 96-well you have seeded? You might want to check some published protocols regarding what supplements different PBMC subsets require. Maybe divide your sample so that you can have conditions that have been optimized for different cells, such as B cells or CD4/CD8 lymphocytes etc.
Seven days is a fairly long time for non-resting cells if no external cytokines/growth factors are added, although this depends on several factors. Also, the medium may need to be changed one or more times, depending on your cell density and the level of cell proliferation.
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