I tried to perform CTL assay with mice splenocytes without seperating them from the stimulator cells. Mice breast cancer cell line (4T1 ) was stably integrated with the target antigen and the cells with mitomycin c treated before they were used as stimulator. After plating in 96 well plate I found there were many stimulator cells present in the spleen cell controls when seen under microscope. Then after 16 h of culture when LDH assay was done we found almost similar OD values in both the test and spleen cell control wells. I used 5000 target cells at 1:80,40 and 20 ratios with spleenocytes. Can anyone suggest how these stimulator cells could be separated fron splenocytes after the stimulation phase?
A few notes first, you are using 4T1 mouse breast cancer cells to activate whole mouse splenocyte immune response in-vitro after 4T1 cells are treated with mitomycin c presumably to see if this pre-treatment makes the cells more susceptible to the immune response or the immune response more effective and you are measuring the resulting cytotoxic response with an LDH assay?
If this is all correct then you don't want to remove the 4T1 cells because the purpose of the LDH assay is to measure the cytosolic Lactate dehydrogenase released by the 4T1 cells that have been damaged by the activated immune cells. You should be running controls including; splenocytes alone, splenocytes + 4T1 cells, splenocytes + mitomycin c treated 4T1 cells, 4T1 cells alone, and 4T1 cells + mitomycin c. This way you will be able to easily determine the source of any aberrant LDH release. You may be seeing similar LDH levels due to too high a concentration of mytomycin c that is overshadowing the cytotoxic immune response.
Apologies if I misunderstood the question or the assay and good luck in future experiments.
You can use gradient density centrifugation on ficoll containing reagents for isolation of lymphocytes from the blood (d=1.08-1.09 for mice, 1.077 for human cells). Viable cells, containing CTLs will be concentrated in interface, whereas dead stimulator cells will be sedimented. I would recommend to use 24-well plates to obtain sufficient numbers of CTLs.
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