Hi Kate,

800.000 cells per well is really to much for 96-well plates. The cells can not receive enouhg oxigen. Normally for proliferation assays you must put 100.000-150.000 cells per well during 5 to 7 days. If you need to put more cells my advise is to use 24 or 48 wells plates.

I will try plating at a variety of densities then.  I want to have a non-proliferative control because I am trying to develop an assay to test antigen response so I am just using ConA right now as a positive control

I agree with the previous comments that 800.000 cells in a 96 well plate are too many. I have worked with 100.000 cells/well with no problem Your cells are probably dying as a result of overcrowding/lack of enough nutrients. Also consider the fact that monocyte derived macrophages are attaching to the plate like one commentator has indicated.

Dear Kate,

I agree with the above comments on cell density, however keep in mind that with PBMCs, you really loose a lot of cells during any PBS washing, handling and throughout the FACS staining, as you also observe. The violet proliferation dye labeling protocol already includes washing steps so I recommend counting the cells after labeling and plate enough cells accordingly.

Hi Kate,

I think the reason that the cell number is low after 4 days is that many of the dead cells have been phagocytosed.   What are the other viable cells left in the culture?  I would guess that you see quite a few with high FSc and high SSc.   Monocytes are very good at  phagocytosing dead cells.   Most cells in the cultures will die without stimulation as noted by Martin, and the dead cell will be phagocytosed by the monocyte/macrophages.  So at the end of 4 days you have some live cells left and some cells that are large as a result of phagocytosing so many dead cells.  These cells might also have a high proliferation dye content.  You will also probably have a substantial number of adherent cells as noted by Kamil and that are likely also to be macrophages and efficient dead cell eaters.Add your answer

I am not an expert in PBMC culture, but I worked with them trying to set up proliferations assays and I had to face different kind of problems.  One of them was the number of cells (in our condition 150 000 to 200 000 are OK). Another one was the stimulus (depends on the antibody clone, concentration, protocol, etc. I suggest to you set up it in your conditions). Other problem can it be the staining (I don´t know when did you count the cells, but usually the staining kills the half of the total number). Another strategy that you can probe is make cultures replicates and mix up at the moment of cytometry or use a bigger plate (48 wells for example). I suggest to you see the cell in phase contrast microscope to check proliferation. Anyway, usually we can´t see the total of cells that we culture, even in short culture protocol, may be you don´t need a lots of events and with 10 000 or something like that is enough.  I wish you luck! Tell us how this story continues.

I will run the assay again with different cell concentrations and different stimuli.  I won't be getting rabbit and mouse blood until next week but I am trying it again today with some pig's blood.

I would be happy to get 10,000 events, I am just baffled that I am only getting 5,000.  Maybe I am just killing my cells and need to work on finding a better cell concentration.

Great! Well, I thought that you were working with human PBMC. Now I understand better. Another thing that you can try is change the culture time (may be 3 days), but sometimes the number of culture days depend on the number of cells . You can also isolate CD3 T cell population, but it is more expensive and may be complicated because you have very few cells. Lucky with the blood!

Hi Kate,

As the others have said it would be worth changing the number of cells that you plate out as 800000 is too many for a 96 well plate. I would go for 100,000-250,000 for that size of plate, in a round bottomed plate to ensure the cells clump together (the T cells will need this for the proliferation). You will always get some cell death in cultures over 3-4 days (especially in your non-stimulated control) and you will also lose cells in the staining process just through the nature of the wash steps. If you need more events you could maybe pool replicate wells to get more events per sample? Anyway, goodluck! Hope the changes you're making help the assay work better!

Hi Kate

As everyone has said- too many cell/well 10e5-5X10e5 at the most.  My advice is to look at the cells in the well under a microscope.  If the cells are proliferating (as they should to ConA) you will see clusters of grapes.  They are easy to see, no need for cell tracker etc.  Look at them every day.  Wells will turn really yellow if they are responding, particularly if you are using RPMI.  Also what medium are you using?   Are you including 5X10e-5 M 2ME.  That is really important for lymphocytes, especially mouse.

ConA proliferation is concentration dependant, think about titering your ConA dose.  It is toxic at high concentration.  The dose response depends on serum concentration.  It will shift to the right at higher serum concentrations.

Right now I am using DMEM with 10% FBS, 1% P/S and L-glutamine, I do not had 2ME.  I've been having some problems with my mouse culture I wonder if it is because I am not adding 2ME.  Do you know the difference between DMEM and RPMI?  I'm the only one in my lab that cultures lymphocytes (others just do MSCs) so I'm trying to optimize cell culture conditions for all my species.

You need to use high glucose DMEM not the standard version.  2 ME is really important for mouse cells.  To set up the assays I suggest using mouse spleen not PBMC, it's easier to get good viable cell just by mechanical disruption.  Check out the Gold Book- Current Protocols in Immunology for detailed instructions.  BTW to isolate lymphocytes from mouse blood you need to use mouse specific ficol-hypaque (we use lympholyte -M)

We currently use high glucose DMEM, but I just ordered some 2-ME, my pig and rabbit lymphocytes seem to be just fine without it but that might explain why my mouse cells were doing so poorly.

I do have a copy of the current protocols and will look into mechanism disruption.  When I isolate my mouse cells I do use a mouse specific ficoll and have good yield from it.