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Is there a way to count lymphocytes from purified spleen using flow cytometry that will give comparable cell # from hemocytometer?
dear Woojong Lee
I usually do this but it might not be 100% accurate. you need to measure the whole sample in your FACS tube and gate on the lymphocytes. then you just calculate the lymphocyte concentration based on these counts and your liquid volume.
But you need to substract the dead volume of the flow cytometer from that volume to get a good result. I test this simply by putting a tube with a cell suspension under the flow cytometer, press start and check how much liquid gets removed form the tube until you see counts. You need to play around a bit, for a BD Calibur it is 120 µl usually.
so you put a sample of the whole spleen cells in 500 µl. then you get your counts in the lympho gate with measurement until the tube is empty. so you know these counts are for 380 µl, you calculate the concentration and get your amount for the whole 500 µl from it.
thats how i did it and it gave quite similar results as a hemocytometer. I think this is likely ok, because with any method you will get a bit other results. If you count the lympho under the microscope it will also differ a bit from the hemoytometer.
dear Woojong Lee
I usually do this but it might not be 100% accurate. you need to measure the whole sample in your FACS tube and gate on the lymphocytes. then you just calculate the lymphocyte concentration based on these counts and your liquid volume.
But you need to substract the dead volume of the flow cytometer from that volume to get a good result. I test this simply by putting a tube with a cell suspension under the flow cytometer, press start and check how much liquid gets removed form the tube until you see counts. You need to play around a bit, for a BD Calibur it is 120 µl usually.
so you put a sample of the whole spleen cells in 500 µl. then you get your counts in the lympho gate with measurement until the tube is empty. so you know these counts are for 380 µl, you calculate the concentration and get your amount for the whole 500 µl from it.
thats how i did it and it gave quite similar results as a hemocytometer. I think this is likely ok, because with any method you will get a bit other results. If you count the lympho under the microscope it will also differ a bit from the hemoytometer.
If you have a cytometer, such as a BD Accuri, then you can determine the cell concentration in a sample, because it measures sample volume along with event (cell) count.
Hello Woojong! You may try gating for the splenocytes. And since you'll probably be getting a percentage of the splenocytes in the population, you may use this to calculate the splenocyte number through cell count (no of events)
Number of splenocytes = %splenocytes * (total event number)
Hope that helped!
Oliver Schmetzer
hi, Oliver,
thank you for sharing your method of doing cell counting with flow cytometry.
But I could not understand some parts.
As you describe, how could you measure the amount of volume removed from the tube? Dose it depends on the speed you choose on the machine to run the samples?
Normally, I could immediately see the counts on the charts, after I pressed the start button. How could you manage that?
Thank you very much for your answer.
Ruibing
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