I am doing mixed lymphocyte reaction for testing the reaction of C57BL/6 splenocytes to Balb/c splenocytes. I cultured the whole splenocytes of Balb/c mice with the irradiated splenocytes of C57/BL6 mice to be used as APCs. I tested for the proliferation of cells using both CFSE and MTT assay. Although I can analyze some proliferation of cells in the form of small peaks using CFSE method but my MTT assay did not work. I did not observe any formazon crystal formation after 5-7 days of culture. Can anybody suggest me what can be the reason for failure for my MTT assay although it worked for my positive control samples (samples stimulated with anti-CD3)?
I've done MLRs for years - though typically BALB anti-C57BL6, using 3H-thymidine, MTT and human MLRs by flow cytometry using CD25 and CD71 expression, but not CSFE.
A couple of thoughts/questions:
1) What dose irradiation did you use? When I was doing it, we used 3000 rad. Either too little or too much can be a problem.
2) Try mitomycin C to treat the stimulators instead of irradiation. When I was trying to make virus-specific CTLs, I had difficulty getting a response with irradiated stimulators, but I could make them with mitomycin C-treated stimulators.
3) When I was at Scripps working with Linda Sherman, we had problems with an unknown disease in the mouse colony that particularly interfered with MLR reactions. Even established CTL clones would become non-responsive when stimulated with affected mice. I never heard if anyone figured out what the source of the problem was, but it did pass with time. The mouse virus ectromelia can interfere with immune responses both in vivo and in vitro, but I don't know if that was the problem at the time.
Hope this helps!
Hi Gary,
Thanks for the information.
I used 3000 rad. Even I could see some lymphoblasts after 4-5 days of culture. But I did MTT assay for this plate on day eight of culture as one of the time point. Is it too long time to do MTT assay for MLR in this experiment? I cultured the plate for 8 days as I found in literature that it usually takes 5-7 days for the proliferation to be observed for the MLR between C57BL/6 and BALB/C.
I would have to dig through old notebooks that are in deep storage to be sure of this, but if I recall correctly, my MLRs were ~ 1 week in culture - 6 days from set-up, pulse with 3H-thymidine overnight.
The MTT assays I have done were also typically 1 week, with MTT added for the final 4 hours.
I do know that if the MLR goes overly long, the CTL go quiescent. Possibly 8 days is too long, but I would be surprized if that were true. Best way to test is to run a time course - set up replicate plates and add MTT to 1 plate on day 4, another on day 5, etc out to day 8 - and see where your peak of proliferation is.
Hello Tabasum,
To be brief and specific. MTT is not an ideal proliferation assay so it's better that you opt for CFSE.
Still if you want to go for MTT.........
tHE REASON FOR NO-FORMAZAN FORMATION IS QUIET SIMPLE, either the cells had died or they haven't proliferated properly. First reason has a large chance of occurance due to contact inhibition of deadherence of cells due to overconfluence. Cell seeding density is also important here. If we start with 5,000 cells/well of a 96 well plate, Ideally we do this for MTT assay of 48-72 hrs incubation but not too long (1week). So you need to do a temporal profiling (as Garry also said) starting with day 3, 5 and then 7. I am sure you will obtain good results and you will also come to know where your cells are entering in a phase of no-division or cell death.
But CFSE is a very ideal assay for cell proliferation and you should focus more on that. Doing MTT is not sufficient for eliminating need for CFSE while doing CFSE can eliminate the need of doing MTT. MTT is more of a cell viability test.
Hope it helps !