xenograft cells harvested from spleen of SCID/NOD mice - (Feb/09/2012 )
I'm use to working with cell lines and this is the first time I am working with xenograft cells harvested from the spleen of SCID/NOD mice. I am currently practicing an Alamar blue cytotoxic assay. I bring the cells up from liquid nitrogen and plate them the afternoon before I intend to incubate them with cytotoxic drugs for 48hrs and then adding the Alamar blue and measure the florescence at 0hr and 6hrs. This is a well established protocol by the lab I am currently in. My problem is that the cells in all of my wells (including controls) die before the day when I'm suppose to add the Alamar blue. I think that maybe its my freeze thaw technique that maybe causing the cells to die very quickly. The only criticisms I am getting is that I am really gentle when I bring the cells up from liquid nitrogen. Can someone please give me some tips on how I could be bringing the cells up successfully from liquid nitrogen or does anyone have any insight on what might be the problem??
How are you determining death? It could be that the cells are just not attaching (presuming they are not suspension cells unlike most splenocytes).
My technique is to remove cells from LN2, thaw quickly in 37 deg waterbath, pipette 1 ml cells gently into 10 ml warm medium. Spin cells down at 100 rcf for 5 min, remove medium, resuspend in more warm medium and plate into appropriate vessel.