I am trying to culture human Monocytes with CD4+T cells for 36hr at 37oC. (following positive magnetic bead isolation of these cell types from PBMC). I am unable to identify the CD14+ monocytes (by flow) following the 36hr culture period. I wonder if someone could help with suggestions on why this might be.
My thoughts on problems/ possible solutions are:
1-should i do negative magnetic bead isolation instead
2- should i change my culture media which is currently AIM V-which contains L-glutamine, streptomycin sulfate at 50 µg/ml, and gentamicin sulfate at 10 µg/ml and 10% human AB serum
3- use accutase to retrieve very likely adherent monocytes
4- cells are currently seeded at 2.5-3x10^5 density in 48 well plate - should I change this, or is any passage required in 36hr incubation period?
5- should i look for differentiation/macrophage markers instead of CD14+ at this 36hr stage
6- ideally i wouldn't shorten 36hr period but maybe I have to, to examine monocytes?
7- are they all just dead?!
Great value/appreciate any help with this problem
Many thanks
Hatty
That is very strange.
1. quick question... the media itself doesn't contain any serum, so i'm going to assume that you add that to it right? Otherwise.. the media should be fine. I wasn't nearly as fancy... I was using DMEM or RPMI with 10% FBS.
2. The density, if it is per well and not per mL... should also be fine. (Otherwise... ideally, you should have at least 5x10e5 cells/mL, I would start at 1x10e6 cells, to ensure good coverage.) The time point is also fine. I have done co cultures well past 72 hours and the markers are all there.
3. CD14 isolation is also fine. and there is no need to differentiate unless that's what you want to look at. The presence of the T cells will automatically cause the monocytes to become macrophages anyway. (Technically just culturing monocytes on plastic will cause them to start to adhere.) So... are you sure you got all the cells off the wells?
4. If your cells are dead, you should be able to detect that by flow. Because the FSC/SSC will be off. And just visually look at the cells... do they look "unhealthy" before you collect them will answer your question.
5. The only other thing is... are you setting your flow correctly?
Shen-An Hwang thank you so much for your response - it is very helpful. I think part of the problem has also been that I am using frozen samples from liquid Nitrogen - apparently the monocytes die quickly after freeze/thaw? have you found this too?
I can see them after magnetic isolation step but not after the culture.
I think a big problem has been that I am not setting the flow correctly as you have suggested - so I am working on that!
I have a final question if that's ok for now - when performing intracellular stainging of monocytes, looking for cytokines, if I have set up an assay with a plate coated in antibodies to stimulate the monocytes, is it then necessary to add LPS as a stimulator, before golgiplug? Can I just use goligiplug without LPS.
Thanks ever so much again for your response
Hatty
If you are using frozen cells, yes, all frozen cells (regardless of what type) will have quite a few die upon thawing and culture. And if you are isolating Cd14 immediately after the thaw... this might not leave you with much, if any, live cells. The rule is almost ALWAYS, if you need to separate your PBMCs, do it BEFORE you put them in liquid nitrogen. Because when you take the cells out, you should really put them immediately into media, otherwise, viabilty can be a problem.
So.. what to do if you have to isolate the CD14 from frozen PBMCs... (I still suggest you try to do this on fresh blood, but if that this not possible...) This is tough because I have never tried it in this order before. But to increase viability, you have to put the cells into media after thawing (make sure you remove the media that the cells were frozen in). Ideally, monocytes should stick to the plastic plates, so you might be able to get a somewhat pure population without Cd14 isolation, and just use adherence. You have to increase the concentration of your cells to at least 1x10e6 cell/mL. The dead cells and the lymphocytes should remain suspended and you can just take that off when you change the media after about 24 hours of "resting"? Again, I'm not sure this will work...
As for the intracellular staining and monocyte activation... what antibodies are you using to "activate" the monocytes? Because it kind of depend on the targets and what the goal of your experiment. Ideally, since you are stimulating this in vitro for 36 hours, there is no need for additional stimulation prior to golgiplug.