Thanks for your help. I am using PHA as my positive control to activate my T cells and look on several surface markers. I have tried once using freshly isolated PBMCs, and it is giving me satisfying viability. So, I am not sure why cryopreserved PBMCs are causing me low viability problem. One thing to notice is my cryopreserved PBMCs are from mouse. Is it possible that mouse PBMCs cannot be cryopreserved like human PBMCs?

 First, you need to sort out whether your problem is with the 48 hour culturing period or with freeze thawing.    Do your mouse PBMCs have good viability if they are freshly isolated and then kept in culture for 48 hours?  Your message above suggests that this is the case.  If so, it sounds like you are having a problem with freezing and thawing. What conditions are you using for freezing and thawing?  How are you storing the frozen cells, in liquid nitrogen or at -80 C?   What medium are you using for freezing?  DMSO concentration?  FBS percentage?  Do you have success routinely freezing, storing, and thawing other cell types?

Hi Robert , thanks for your help. Yes, I obtained better viability if I used fresh isolated samples. I cryopreserved PBMCs in 90% FBS and 10% DMSO. I normally store them overnight at -80 C, then transfer to liquid nitrogen tank. After the thawing process, they normally gave me high recovery (about 90% viability using trypan blue exclusion method). But when I cultured them, it seemed like these samples just died and gave me very low viability. I have not tried with other cell types before, as my experiment deals only with PBMCs.  I am not sure what is actually going wrong. 

It sounds like your freezing conditions are generally okay. However there are a variety of subtle conditions that can cause problems during freeze thawing. For example:

-- thaw at 37C with agitation.  Lower temps without agitation will give decreased viability.  Too much agitation is also bad.

-- When you freeze, the cell concentration should be below 3E7/ml.

-- freeze in a freezing block to reduce ice crystal formation.

-- Use low speed spins to remove DMSO after thawing.  High speed damages cells in DMSO.  

-- Remove DMSO immediately after thawing.  It must be removed quickly in sensitive cell types.  

Cell damage from freeze thaw often does not show up for a day or two, when viability will drop (which is what you are seeing).  This why it would be good idea to test your freeze thaw technique on another cell type like HL60 or H9 cells, just to rule out freeze thaw damage.

Robert, do you mind to share with me your thawing protocol? I normally thaw my PBMCs with gentle agitation at 37 C water bath, add 1mL of warm cRPMI dropwise to 1mL of cryopreserved PBMCs in cryovial, transfer to centrifuge tube and add 8mL cRPMI, spin at 1200rpm for 10 mins, wash another time with cRPMI and count the cell. Does this sound fine to you? 

That protocol sounds good assuming that cRPMI contains at least 10% FBS and that 1200 rpm corresponds to no more than 600 xg and the spin is done at room temp or lower.  If you can get good sedimentation in 5 min, then use this shorter centrifugation time.  If the pellet is too compressed, cells can be damaged when resuspended.  Resuspend by tapping or flicking the tube with your finger before adding fresh cRPMI or by gentle pipetting with a large bore pipet or micropipette after adding RPMI.

Robert, really thanks for your help. Hopefully I will get a good viability for 48 hours stimulation after using a more stringent protocol. 

Hi Jia, if you're interested in looking at T cells, I'd suggest adding some IL-2 to cell culture at the concentration of at least 250 U/ml. 

Hi Paulina, yes, I'm looking at T cells. I freshly isolated mouse PBMCs, let them rest for 12 hours, gave PMA treatment and cultured them for another 48 hours. The one with just media alone gave me ~40% viability, while with PMA added gave me lower viability as PMA concentration increased. Is this viability fine? Will adding IL-2 help to increase the viability of T cells more? Thanks!

Low concentrations of IL-2 will definitely help keeping the cells alive without stimulating them too much. PMA stimulations are tricky - too much for too long will certainly cause activation-induced cell death, which is probably what you are observing. I don't know what your experimental question is, but to check for up/down-regulation of surface markers, overnight stimulation would be more than sufficient to notice any differences. 

Thanks a lot Paulina, I will add IL-2 next time and see. The viability rate of PBMCs with media alone (no stimulation) is about 40% after 2 days, is this considered fine? I am stimulating my PBMCs with different mitogens (PMA or PHA) in order to find one that can activate my T cells and up-regulate my surface markers, so that I can use the result as my positive control. Many papers indicate that they activate T cells for 48 hours to express my markers, that is why I am trying with 48 hours. 

Jia, at what concentration do you seed your cells? Also, do you use a flask or a plate? 

Paulina, the cell concentration was 1million cells/mL. I cultured 500uL of them into 24 wells plate. 

Your cell concentration may be a bit to high. I'd target it at 0.5 million/ml. You can continue using 24wp or use U-bottom 96wp (105 cells/200ul/well). I think you should be able to get a good viability now. Also please remember you're culturing PBMCs under T cell condition. Other cell types could be dying. Best way of assessing T cell viability would be via flow cytometry. Gate on CD3+ and compare 

Thanks, Paulina. I did try with several cell concentrations, and as you have mentioned, 0.5 million - 1 million/mL would always give me the best viability . I have been using BD FVS 510 to check the viability of all events. I initially thought that since PBMCs have about 75% T cells, so the viability rate should be around 70% after culturing, but it seems like they don't work in this way. Viability is normally low after stimulation, and about 60-80% of viable events are expressing CD3+.  T cell viability is hard to be determined in my case because my PBMCs that are dead will bind non-specifically to the antibodies. That is why I have started to include a viability dye to exclude all these signals from dead cells. If I gate on CD3+ population first and then check the viability of them, it is very likely that what I have gated is the combination of real CD3+ population, together with non-specific bindings contributed by other dead PBMCs. 

Hi Jia, another neat way of assessing viability using flow cytometry is staining your PBMCs with CFSE. Make sure your cells are well-rested after thawing (overnight is enough) and you have enough cells to stain as loosing 20-30% of cells isn't uncommon. At the time of reading, you can gate CFSE+ -> CD3+ -> viability dye. That should give you a very good estimate of the condition of your cells.

Also, prolonged stimulation with PMA will cause activation-induced cell death. You may want to choose concentrations at the lower end of spectrum, or decrease stimulation time to 24h. 

Hi Paulina, sorry for late reply. Thanks for your suggestions. Yes, I realize a significant portion of my PBMCs will die after resting overnight. Is it always advisable to rest my PBMCs overnight, and only stimulate them with PHA or PMA? I will just waste a lot of my cells, and I only have limited PBMCs from each mouse to play with. 

I certainly have to find a way to increase my viability. So far what I have noticed is that the stimulated PBMCs will always have a lower viability than that of the unstimulated one. I just hope that the viability does not go too low in both unstimulated and stimulated samples, so that I can record enough viable events and do analysis on them. 

Hi Jia, no worries. Hope you had a great weekend.