I'm trying to optimize an LDH cytotoxicity assay (https://www.takarabio.com/products/cell-biology-assays/cell-biology-reagents/ldh-cytotoxicity) but am running into some issues. I'm using NK92s as my effector cells and K562s as my target. Both are highly viable before starting the assay (>90%). I'm using the recommended number of target cells as specified in the kit's manual (1x10^4) and looking at E:T ratios of 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1. Culture is in RPMI + 1% FBS for 4h at 37C.
My effector-only controls (wells that only contain NK92s) are very close to my effector+target wells for both ratios. I've checked cell viability and by the end of the assay, the NK92s are at ~85% live. When I calculate % cytotoxicity using (E:T - E - Spontaneous) / (Max - Spontaneous), I get a negative number. I've included a picture below showing some sample data with 10:1 and 1:1 ratios.
Any ideas why this assay isn't working? Is it possible that my NK92s are just not killing anything? Thanks all!
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