Lymphocytes do die in cell culture due to activation-induced cell death. If you need long-term culture, change media every 2-3 days. ACK lysis buffer should be fine if you wash the cells after the lysis.

What's the final concentration of ConA added in your cell culture ? I routenily use this kind of assay for veterinary species and it's true that the mitogen concentration used is crucial.

I am guessing that your COnA concentration is too high. This causes Overactivation of your lymphocytes and eventually apoptosis. I would decrease con a concentration or used a milder mitogen. In addition,  your 7 day culture could be too long.

I use 5ug/mL of ConA, I checked on day 3 and they were beginning to die so I don't think it was the length of culture time because they should be alive by then.  What confuses me is that I don't see proliferation peaks when I look at the CellTrace Violet channel which makes me think that it isn't AICD.  The one CTV peak MFI did drop from around 10^4 to around 10^3, but I had just assumed it was because they had died, but I guess it could also mean that they all proliferated at the same rate and died.  For my pig cells I get very nice sharp peaks at 5ug/mL of ConA or PHA and my rabbit cells don't really proliferate at all so I know there is some species variation.

The only thing that doesn't explain is that both my unstimulated cells and my unstained/unstimulated cells are still dying and their CTV MFI has dropped as well so it can't just be the ConA.  I've attached pictures of the fsc/ssc for day1/3/7, these are representative of both the stimulated and unstimulated.

Does this happen for anyone who cultures post-lysis?

You probably also need IL-2

Adding IL-2 still wouldn't explain why both the unstimulated and stimulated cells are dying.  I believe that mouse lymphocytes can sit in culture for at least a week with no stimulation without dying, I'm not trying to passage them for any length of time.

Hi Kate

From your plots, it appears that your buffer is indeed lysing your cells. Some manufactures require that the ACK RBC lysis buffer is diluted with deionized water prior to use, while others sell it ready to use.  

We use the following protocol from BioLegend for human PBMCs

1. Dilute the 10X RBC Lysis Buffer to 1X working concentration with deionized water. Warm the 1X solution to room temperature prior to use.

2. Add 2.0 ml of 1X RBC Lysis Buffer to each tube containing up to 100 µl of whole blood.

3. Gently vortex each tube immediately after adding the lysing solution. Incubate at room temperature, protected from light, for 10-15 minutes.

4. Centrifuge 350 x g for 5 minutes. Aspirate supernatant

5. Resuspend the pellet in PBS, wash.

6. Resuspend and proceed with further procedure

I am using Invitrogen's ACK lysis solution that should be at a 1X from the bottle, I will dilute it and see if it helps, the manufacturer does not recommend it to be diluted.

I also quench my reaction with 1% FBS so I would hope that the osmotic gradient would be removed post-lysis.

Well, Heriberto, I do not think that the plots suggest lysis, since the cells grow smaller (suggesting hypertonic, not hypotonic environment) and more granular on the scatters (suggesting apoptosis). Moreover, I would not expect lysis after 3 days in a cultivation media (middle plot) - the cells were washed post lysis and transferred into culture media right? I lean toward apoptotic death either by negligence (lack of stimulation) or by overstimulation (activated cells clear themselves from the system within days, to minimize collateral damage). So the cytokine mix and their concentrations need to be balanced and nutrition maintained according to the number of cultured cells (too low concentrations of cells sometimes cause death as well as high concentrations - this contributes to the lack of - or over- stimulation). Unfortunately, there seem to be too many factors to suggest one quick solution to this problem.

Kate

Have you tried using Lymphocyte-M? It similar to Ficoll (which i use for human blood only) but it gives a higher yield of viable mouse cells post processing. I don't use any lysis because its very harsh on the cells and it results in poor surface staining. If you have a chance compare a lysis vs Lymp-M treatment using your side and forward scatter and you will see a world of a difference. 

Karen

I would not recommend to dilute the buffer, because red cells lysis would not be effective, even under non-stimulated conditions cell death should not be at the percentages shown by your plots, especially at day 1 and 3. As Jan indicates, there are many factors to be considered. Have you counted your cells after lysis? If you stain them with tripan blue and count them with an hemocytometer, immediately after lysis procedure,  you will know if the buffer is affecting the lymphocytes. 

I do not see significant cell death post-lysis using a trypan blue stain which is why I am confused about their death.  I had assumed it was the lysis treatment because both my stimulated and unstimulated cells were dying, but it could be that they are dying due other some other factors.

The reason I don't use a ficoll-like separation is because I only get between 400-600uL of blood and it's always been very difficult for me to get any good yield from it.  I've never tried lymp-M, which company sells it?

I use an automated coulter counter to get my cell density to determine how many cells I want to plate in each well (I aim to plate 200,000 but sometimes it's a little lower because I never get more than 5million lymphocytes from the 400-600uL of blood) but I've always been suspicious that the cell count has been off for the mouse blood because it usually has some amount of contamination with RBCs (which is why it is difficult to count cells on the hemocytometer because the RBCs are difficult to differentiate from the lymphocytes without a diff-quik stain) that gives me an artifically high number, so it could be that I am plating too few lymphocytes and they are just dying because of that.  I did not know that lymphocytes would die in the span of 2-3 days in culture if there were too few of them.

What concentration would you recommend to plate them at?

I should also add that the media (DMEM) color changes slightly - from a bright pink to a more dull pink but is nowhere near yellow like I would imagine to see for highly proliferating cells.

Hi Kate,

Most protocols suggest cell concentrations between (1.5 x 106-3x 106 cells/ml), but you would have to determine which one is best for you. Have you seen your non-stimulated and stimulated cells under the microscope? If you see a dull pink media, it could be because of a high concentration of cells or debris. T cell blasts should be evident by the second day, also a lower pH.

So on Friday I separated half of my cells through lysis and the other half through ficoll gradient and today I took out a sample of each and tested them with trypan blue and found that 50% of the cell in the lysis wells were dead compared to 1-5% in the ficoll gradient.  Now the yield was a lot lower than I'd like it to be, does anyone have a good protocol for using 1.077 ficoll to separate out mouse blood?

 I use Gey's solution for lysis, in theory is more gentle with lymphocytes.

then, maybe you are using too much ConA?

also the staining with cell tracers can kill some cells. you could play with the concentration or timing of CTV staining.

I've done it many times and not had a problem. ACK is fairly innocuous to mouse lymphocytes since they have the appropriate channels to rectify ionic balance. You may wish to try a bit of IL2 in your medium, especially if your concentration is low. A silly question: are you using a stock ACK and diluting? If so, perhaps the problem is your water? I'm shooting in dark, since I've never had this problem...

Your plots suggest that you don't have a problem with the buffer. That is because if your buffer was the problem, the lysis of the cells occurs in a few minutes, and you should be able to see it already on Day 1. I think that the problem is that your stimulated cells die because they get only signal 1 through conA (you need co-stimulation and/or IL-2). Your unstimulated cells die, because they need IL-2 as a survival factor. For both your samples, you need to add IL-2 to keep them alive. Also, you can try RPMI based medium supplemented with human serum (in case your lymphocytes are from human blood).

Victoria,  I find this strange because my other species do just find without IL-2 added to the culture and will stimulate well with ConA or PHA alone.  I've also never heard of a protocol that requires IL-2 addition with ConA to work, I've heard it just being used on its own.

Hello Kate, I encountered exactly the same problem.

We use RBC lysis (BD Pharmlyse, 3 min followed by 2 washes) for isolation of mouse BM cells for transplantation. We checked viability after isolation (right before transplantation) with 7AAD and it is over 95%. Also transplanted cells are functional since we get chimerism after transplantation.

Now we used the same protocol on spleenocytes and again viability after isolation is fine. We used some of these cells in proliferation assay and positive controls (stimulated with PHA) responded well though I got a feeling that the number of cells in the wells was smaller on day 3 and 5 of culture as it was on the beginning (so some cells are dying and degrading??).

I also put some of the isolated splenocytes into RPMI medium with FBS and left them in the CO2 incubator overnight and measured apoptosis and cell viability the next day. I noticed the same population of dead cells you are talking about. Aproximately 1/2 of the cells were dead, I dont really see a substantial percentage of apoptotic cells. I  kept some of the same cells in the fridge after lysis and I checked the viability the next day and it was fine - no second population left and up from lymphocytes.

My reason for using RBC lysis is the same as yours - I just dont get enough cells with ficoll. So now I dont know what to think and if cell lysis is ok for removal of RBCs or should I avoid it.

Does anyone know if IL-2 is required to maintain mouse cells in culture?  I am following a protocol form current protocols in immunology and they just recommend using ConA (as long as there are APCs in culture and mine does have APCs) and no mention of IL-2, I would suspect that the cells would produce their own to maintain?

I always use IL-2. They'll start to produce their own but I find it usually takes a couple of days. I usually just add it to a final of 30 U/mL

 Thank you Govinda, do you know if this will induce proliferation in my lymphocytes, I want my unstimulated cells to proliferate so they can act as a negative control.

To actually induce proliferation of T-cells in the absence of any stimulus (ie. antigen, agonistic antibodies, mitogen, or pharmocological agents like PMA/Ionomycin), you'd need a huge dose of IL-2 (on the order of 6000 IU/mL). T-cells that are expanded this way usually behave pretty differently regarding their proliferation and effector characteristics though so you'd have to think about whether it's an appropriate control for your application.

Did you get any resolution on this? I've been comparing isolation of lymphocytes from 200 ul aliquots of mouse blood using EDTA as the anticoagulant. ACK appears to kill a large number of lymphocytes, unless I do a very short incubation and a very large volume of HBSS to wash. FicollPaque gives higher viability. I add 1.8 ml of HBSS to the blood, underlay 3 ml of FicollPaquePlus, spin 30 min at RT using 15 ml conical tubes, and get a decent buffy coat layer. I still have trouble consistently sucking off that layer, but the yields are better than ACK. The CD8/CD4 ratio differs between ACK processed cells versus Ficoll, and I'm still trying to figure out what's going on there. I haven't tried culturing them after isolation.