I am working with tonsillar B cells from patients. A major problem is that it is very difficult to coordinate the flow cytometry time at the flow core facility with the time when the patient samples come in. Most of the time I have to freeze the cells and thaw them at some other time for flow sort. The biggest problem is that after a freeze/thaw cycle, the cell viability drops substantially. For instance, I got 81% viability from the mononuclear cells from the most recent patient sample. However after a freeze/thaw cycle, hardly 10% were alive, which is devastating. Sometimes when I am lucky I could get about 30-40% viability after thawing.
We are in the process of disscussing with the core facility staff about sorting for us after hours but there is always limitation. I freeze the cells in 10% DMSO, 90% FBS. I use isopropanol insulation box. Cells are thawed in 37 degree wather bath quickly and resuspended in cold complete media. I wonder if anybody has gone through a similar situation and if there is anything better way to up the cell viability cells when you have to freeze them?
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Posted 01 September 2009 - 10:45 AM
TracyDuke, on Aug 28 2009, 03:54 PM, said:
I am working with tonsillar B cells from patients. A major problem is that it is very difficult to coordinate the flow cytometry time at the flow core facility with the time when the patient samples come in. Most of the time I have to freeze the cells and thaw them at some other time for flow sort. The biggest problem is that after a freeze/thaw cycle, the cell viability drops substantially. For instance, I got 81% viability from the mononuclear cells from the most recent patient sample. However after a freeze/thaw cycle, hardly 10% were alive, which is devastating. Sometimes when I am lucky I could get about 30-40% viability after thawing.
We are in the process of disscussing with the core facility staff about sorting for us after hours but there is always limitation. I freeze the cells in 10% DMSO, 90% FBS. I use isopropanol insulation box. Cells are thawed in 37 degree wather bath quickly and resuspended in cold complete media. I wonder if anybody has gone through a similar situation and if there is anything better way to up the cell viability cells when you have to freeze them?
We are in the process of disscussing with the core facility staff about sorting for us after hours but there is always limitation. I freeze the cells in 10% DMSO, 90% FBS. I use isopropanol insulation box. Cells are thawed in 37 degree wather bath quickly and resuspended in cold complete media. I wonder if anybody has gone through a similar situation and if there is anything better way to up the cell viability cells when you have to freeze them?
Why don't you try to resuspend slowly in warm medium?
