Chromium Release Assay - Troubleshoot (Aug/12/2008 )

I have been trying to do cytotoxicity assay without much success. I do all steps really carefully but the results are not good.

I have 4 different assays that I do at the same time - triplicates of each but the SEM is very high. What could be cause of such inconsistency?

Any tips and tricks or any guidance.

What technique do you use to measure chromium? It is incredibly easy to contaminate samples during the collection process.

At low concentrations, it is harder to get tight CVs. What are your CVs at the middle and upper end of your standards?

I think it should be contamination with the live cells.

I collect (from total 200ul) 100ul of supernatant really carefully not to contaminate and read them in gamma counter.

One step I am not sure of if I am doing right. I use U bottom 96 well plate to do the assay and after all the steps are over, I centrifuge the plate @400 rpm for 1 min (as suggested by senior) to pellet the lymphocytes (target cells) to the bottom. If the spinning is not enough then it should be hanging in the supernatant also contaminating.

hi,

do you use Lumaplate?
how long do you incubate cells?
it is nice to centrifuge plates before collecting SN, but not always necessary if you take care... We use instead of U V bottom plate
is the signal in the positive wells high? and is SEM for negative control as high as for test wells?
it could also come from a non homogeneous distribution of any of the target/killer/additive in each of the wells...

Tryptofan, seeing you after a long time.

I completed the assay and the results were good. I had made mistake in the number of target cells I had been using. It was too small than one I had done on my pilots. I corrected them and it looks fine now. But, I still have to show it to my supervisor; if he does not like it I will have to repeat.

I use the 96 well U bottom plates. I don't know if they are Lumaplates. I guess not if these are the Lumaplates.

What are the advantages of using these plates and is the result significantly different if we use the tissue culture 96 well U bottom plates?

I incubate 30 mins with serum @ 4 deg C and 45 mins with rabbit complement @ 37 deg C. What do U suggest?

I will check SEM of the negative controls and follow.