2 replies to this topic

[corduroy]

Hey all, I hope someone can give me a little guidance here.
Here's the scenario. Let's say I have 200k cells per ml in wells. Sample A is the control and Sample B is treated with a known substance that kills 90% of the cells at 48 hours. When I run flow at 48 hours, do I: use the same volume as the control to read or do I count out the same number of cells between the control and treated samples and read those samples (which would be 10x of the treated sample)? Readings would be done on PI or 7aad and several antibodies for expression.

I have a few thoughts but I keep on going back and forth on this and am unsure. If I'm not too clear, let me know - it's something I'm working on Thanks.

[CellSpecific.com]

If what you're after is "% of cells dead or alive" after treatment, there is no need to count the cells prior to running the samples. You might even want to collect the same number of events but this should not affect the frequency of dead or live cells. Either way the frequency of live or dead will remain the same for either sample. E.g., Untreated - 2 % dead/98% live; Treated - 90% dead/10% live.

If you need to present the data in absolute numbers and since you know the absolute number of total cells/sample ~200ul/well = 40,000 cells. Just multiply this amount with the frequency of dead or live cells to acquire the respective absolute values. Hope this helps you.

Edited by CellSpecific.com, 27 October 2009 - 07:27 AM.

[canotto]

corduroy, on Oct 27 2009, 04:04 PM, said:

Hey all, I hope someone can give me a little guidance here.
Here's the scenario. Let's say I have 200k cells per ml in wells. Sample A is the control and Sample B is treated with a known substance that kills 90% of the cells at 48 hours. When I run flow at 48 hours, do I: use the same volume as the control to read or do I count out the same number of cells between the control and treated samples and read those samples (which would be 10x of the treated sample)? Readings would be done on PI or 7aad and several antibodies for expression.

I have a few thoughts but I keep on going back and forth on this and am unsure. If I'm not too clear, let me know - it's something I'm working on Thanks.

I think that you will find an answer to most of your questions regarding this issue in the following reference: King et al,Detection of dead cells and measurement of cell killing by flow cytometry, Journal of Immunological Methods, 2000