I need to check for the MLR (Mixed lymphocyte reaction) for the particular cells using MTT assay. I have to co-culture the responder cells (CD8 T cells) from one mouse strain with the stimulator cells (irradiated APCs) from the another mouse strain. Since I am planning to measure it by MTT assay, what are the important controls which I should use? Also, after measuring absorbance at 570 nm, how should I interpret the results from that, i.e. how should I determine the % viability of each sample?
You car evaluate lymphocite proliferation using MTT and other similar products as Roche Cell Proliferation Reagent WST-1, a water soluble derivate of MTT (yellow color) than you can read as a ELISA at 450 nm.WST-1 is more sensible.
You can see our protocols in two of our publications, that you can download from reseachgate:
For MTT: Serrano, A et al. Arch Biochem Biophys. 1998 Feb 1;350(1):49-54.
For WST-1: J Immunol Methods. 2014 Jan 31;403(1-2).
Lucky
A.
Dear Tabasum, I would not recommend to use MTT for evaluation of MLR results. To the end of culturing, MLR cultures contain debris from dead (non-activated and stimulator) cells, which result in marked scattering of light ray. You can compensate this by subtraction of OD measured at 690 nm, but activated lymphocytes transform MTT weakly in contrast to macrophages or in vitro cultured tumor cells. As result, you will see only small difference between formation of MTT-formazan in cultures stimulated with syngeneic and allogeneic stimulators. It would be much better to use measuring of 3H-thymidine incorporation. Good results may be obtained also with CFSE staining of responder cells and subsequent analysis by FACS.
Hi all,
I did not observe any proliferation after setting up the mixed lymphocyte culture between C57BL/6 and BALB/c cells. I had set up the culture for purified CD4 and CD8 T cells, respectively isolated from BALB/c mouse mixed with irradiated splenocytes (2500 rad) isolated from C57BL/6 mouse using different cell ratios. However, after 3 day culture, I did not observe any lymphoblasts in the culture and even after adding MTT, there was no farmazon crystal formation. Since by mistake I had not kept any positive control (like anti-CD3 stimulated cells, I don't understand what might have gone wrong. Earlier I tried the pilot experiment with different concentrations of anti-CD3, the MTT assay worked well. Please advice if I need to add up something.
What method did you use to isolate purified CD4 and CD8 T lymphocytes?
Positive or negative selection? If positive, you could obtain inhibitory effect due to blocking coreceptors.
Using positive selection you can isolate subpopulations of T cells, but their capability to respond in MLR will be compromised due to blocking of coreceptor by mAbs.
MTT can measure cell proliferation but not viability. Parameters critical to a MLR include, number of responding cells per well (150,000 works for us), ratio of stimulators to responders (1:3 to 1:10 is likely optimal) and time of thymidine addition, which varies with species. I might note duration of co-culture with thymidine (I like overnight) is impoatant. As pointed out MTT is supoptimal, although it can be made to work. However, the stimulation index (SI) is generally low relative to the use of thymidine. You don't have to isolate T-cells or CD4 or CD8s to make it work. Note, you can use a one way MLR, where you irradiate the stimulators (most common) or a two way MLR (where both lymphocytes respond). The one way MLR is most common and is easier to understand your results.
I need to avoid mouse cells and do not have access to human donors. Could someone please suggest me an alternative source for the lymphocyte cells to be used in MLR
Murine spleen cells from two different strains. For example irradiated C57Bl6 (simulator) and non-irradiated (responder) from Balb/c spleen cells.
Response to Dipti:
To make an MLR you need use donor-cells and recipient-cells of the same specie (ice/mice or human/huma). A typical reaction involves MHC-mediated Tcell activation.
Xenogenic MHC recognition is generally nonexistent or, if anything, very inefficient.
Best regards:
A.
Response to Dipti:
You can use any species. I have done MLRs on Mice, Humans, Primates, Cats, Dogs, Cattle, Horses, Mink, etc. As long as the animal is out bred all you need is two blood donors. Note, you could use a cell line as your stimulator cells. Just irradiate. Might need to sort out the optimal R:S ratio.
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