I will be doing experiments involving culturing and stimulating PBMCs and harvesting them 24 hours later. Basically, they will only be in culture for a day. I am interested in a potential secondary effect which may differ in B cells vs monocytes, so after this treatment I will have to isolate the B cells and monocytes from each treatment for qPCR. My question is, what kind of plate should I use? If I use round bottom, I would have to isolate monocytes first via CD14 positive selection and then isolate the B cells via CD19 negative selection. Or would it be better to use a flat bottom plate, isolate the B cells via negative selection, and have the remaining adherent monocytes. Or is there a 3rd option which is better? What is the best way to do this? And how many cells/well would be appropriate?
Please see this Blood paper (which cites some methods in primary sources) on B cell isolation. The group specializes in B lymphocyte biology.
http://www.bloodjournal.org/content/112/6/2554?sso-checked=true
In this PBMC assay (total PBMC), the density used was 1x10^6 cells/mL in RMPI-C. The volume is 250 uL. See p17 of the PDF below.
http://ntp.niehs.nih.gov/iccvam/docs/protocols/pyro-pbmcil6.pdf
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Hope this helps somewhat. Good luck on your exp.!
We have found that separating cells after stimulation is particularly challenging because activated cells usually express higher levels of adhesion molecules and have a tendency to clump together. Even using 5mM EDTA is often insufficient to dissociate these clumps and trypsin or accutase treatment may affect your cell marker-dependent isolation. Thus, expect lower purity than should be expected from commercial cell isolation kits.
If you stimulate PBMCs, monocytes will probably receive differentiation cues which will lead to strong attachment to plastic and differentiation to macrophages. This will likely not happen in your unstimulated controls. You will thus have important differences in monocyte yield between controls and stimulated wells which you should take into account. You may want to use ultra-low attachment plates (from Corning, these cost a lot) to limit these adhesion effects. CD14 expression is usually reduced once monocytes differentiate to MDM so that CD14 positive isolation will maybe not work as well.
As you can see, what you want to do is far from simple but I think it can be done with a little bit of optimization. Good luck with your research!
thanks for the suggestions! I think I will forget tissue culture treated plates and just use petri dishes. With those the monocytes will not adhere and cell isolation should be easier. What do you guys think? Or maybe culture the PBMCs in 50ml polypropylene Falcon tubes, since the cells will all be in close contact and the monocytes should not adhere to polypropylene?
I had tried culturing a mouse monocytic macrophage line RAW264.7 cells in petri dishes, in which case they did behave more like suspension cells (rather than their normal adherent form in coated culture dishes). However, although they also did proliferate over time, their morphology was not good, and in a test on phagocyte oxidative burst, the cells cultured in petri dishes performed poorly. So I gave up on culturing them in non-adherent form. You may try if it fits your experimental purpose, and if you figure out ways to optimize culture, like throwing in suitable supplements to ensure healthy growth.
In culturing in a Falcon, you need to resolve the aeration and waste/nutrient distribution issues.
I found using flat bottomed culture plates to work best for me ...small petri dishes were beneficial for harvesting maximal numbers of cells.
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