I am new to flow cytometry and have not had formal training on setting appropriate voltage / (gain?) values. I ran PBMCs isolated from whole blood, and tried to adjust FSC and SSC values so all my cells showed on the FACS plot... but I believe I must have done something incorrect as I dont see distinguishable populations of monocytes and lymphocytes, as I believe I should
(I also stained for CD45 and CD14, so I should theoretically have a double positive and single positive population when I gate, which I do not)
Any advice as to what I may have done wrong would be much appreciated!
I attached an example of my FSC-A vs SSC-A so you can see how it looks..
Hi Margarette,
We usually view our FSC and SSC in linear scale, so that may be the reason you SSC is pushed up so high. Also, it looks like your voltages may have been set too high for your FSC and it is cutting off your monocyte population. I suspect you will only have one population that is CD45+ from the looks of it.
When you set these voltages do you use a sample or control? We usually set voltages while acquiring for compensation so we are able to see the FSC/SSC plot and adjust accordingly. Then we go through voltages for each other parameter. Hope that is helpful.
Hi Margarette,
We usually view our FSC and SSC in linear scale, so that may be the reason you SSC is pushed up so high. Also, it looks like your voltages may have been set too high for your FSC and it is cutting off your monocyte population. I suspect you will only have one population that is CD45+ from the looks of it.
When you set these voltages do you use a sample or control? We usually set voltages while acquiring for compensation so we are able to see the FSC/SSC plot and adjust accordingly. Then we go through voltages for each other parameter. Hope that is helpful.
Hi Erik,
Okay, yes, it does appear that one population is CD45+ CD14-, so that may be the lymphocytes. I did comps with beads, so the FSC and SSC was pretty different. BUt, my first sample I ran was unstained PBMC cells, which I tried to use to readjust my FSC SSC voltages before running my samples. However, I was having difficulty getting it to look like both populations were on the plot. Perhaps FSC needed to be lower, but it was already less than 300, and I was told it should optimally be 400-600 range. Perhaps even lower would be the way to go, for next time?
Thanks for your response!
Personally I have heard of an optimal voltage range for fluorochrome channels which is generally 400-600 as you said, but should be of course determine for each cytometer individually. I usually try to set my lymphocyte population around 80k-100k, but if I will be looking at all PBMC populations then I will also make sure everything is contained in the plot. You can perform singlet gating, live, & CD45+ gating prior to your FSC/SSC gate to clean up the debris and dead cells to ID your populations too.
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