CTL LDH assay kit..... Too much of spontaneous lysis?! - (Jun/11/2002 )

Dear Netters....
I am trying to standarise the LDH based CTL assay kit from Roche. I'm using p815 cells as my targets for lysis with C57 sleenocytes primed in vivo by the same cells. My problem is I'm getting negative values as the spontaneous release is so high. My spontaneous release is as high as 50% of the Maximal lysis. Is anybody out there facing the same problem? It would be of great help if you could give me some hints.
I have tried checking for various conc of BSA(1-5%) versus FCS(1-5%), different media, different cells as I thougth that I should rule out that being a problem with p815 exclusively. But irrespective of what cells I incubate with assay medium alone ( no effectors) and check for LDH- it turns red like mad. Does it mean that I HAVE to do Cr51 release and give up on the LDH kit? Please reply.

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i dont know about the assay you are doing but we routinely use the Cr51 CTL assay and it works really well. You may have to do this type of assay insted, its pretty easy to do.  We use EL4 cells (Thymoma cell line that is Kb restricted) to do our T cell killing assays with.
Spontaneous release often is high if your media is wrong, or the target cells grow crappy. Also it may be a technique thing like somehow not washed the cells of excess label.
Hope this is of a tiny bit of help.
Maybe try peptide pulsed targets?

Regards

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Hi Richard,
It sounds like you know a lot about Cr51 CTL which I am trying to set up. On ocasion I have high Spont. release and repeat it again but my problem is that my quadruplicates are mostly not reliable meaning that from 4 replicates 1 or 2 are out, I use Scatron collection system but I push the sponges soaked with Cr51 into the regular tubes for Cobra counter. Do you use the same system or not?
I work for Columbia University Pediatric Cancer Research and I need to make it work reliably. I will appreciate your help. Maybe you can send me your procedure
ls18@columbia.edu
Thanks,
Luba

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