Hi everyone,
I am currently doing NK92 cytotoxic assay and I found a problem that NK92 are likely to form clusters themselves very quickly and spontaneously. I think this might affect their killing efficiency because not every cell has sufficient change to get in touch with their target cells.What's more, I think there must be some treatment before injection of NK92 into human vessels to prevent their cluster formation and thus avoid thrombus formation. I tried to irradiate NK92 at dose of 10Gy for 6 minutes and they quickly form clusters when suspended in αMEM. Did anyone has the same experience and solutions? Thank you
Happy and healthy human NK cells like to grow in clusters, but this shouldn't affect your functional assays as the washing and counting steps involved in preparing the NK92 cells for use in the assay will disrupt these clusters and when they reform the target cells will be incorporated (if they're suspension cells). If you're not getting good levels of killing in your assays then remember that NK tumour cell lines almost never kill as well as primary human NK cells. There are two obvous tricks that you can try to improve killing: 1) increase the ratio of effectors to targets. 2) treat the aliquot of NK92 cells that you're going to use in the assay with 50U/ml of IL-2 the night before you perform the experiment.
Agreed - NK92 are their healthiest when clustered. We've never tried to break them up for killing assays, but I've noticed that it doesn't take much force to separate them (gentle pipetting will do it), so if you have access to a shaking incubator, I would suggest trying that. Once you've got your assay set up and your effectors and targets co-cultured, try dissociating them with a pipette, then quickly putting them on a shaker plate and rock them gently throughout the procedure. Good luck!
Beste you ask one of the persons that developed this cell-line, which is Professor Torsten Tonn in Dresden / Germany [email protected]
You can try using very low concentrations of beta mercaptoethanol.
I echo the sentiments expressed above: culture with IL-2, try increasing effector to target ratio, they're healthiest when they cluster, and we also culture in low concentration beta-ME. Further, you can perform your cytotoxicity assays in V-bottom plates and at the start of the assay, after you have thoroughly mixed the cells to break up clumps initially, you can spin the plate at low rpm to bring the cells together. This way, the assay starts out with the max effector:target contact. I also echo the thoughts of others about injection: NK92 in clusters shouldn't affect their activity in vivo either.
Hi, I totally agree that happy and healthy NK92 cells grow in clumps. It makes me worry if they don't. I have them now in culture at the moment (I always culture them with 200 U IL-2/ mL) and I just performed functional assay. They were alive and functioning properly. Before my assays, I always resuspend them with pipette to make them loose. Good luck!
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