Hello everyone, I’m planning to perform flow cytometry and have a few questions. When doing flow cytometry, do all the controls for fluorescence compensation (such as single-stained controls and unstained controls) need to be prepared within a single experiment?
In my case, I am working with MSCs and using CD105+, CD73+, CD31−, and Live/Dead. However, since CD31 expression is only about 1%, I would need a very large number of cells to secure enough positive events.
This means harvesting cells from many flasks, which is quite complicated and labor-intensive. I would really appreciate any advice!
Hello Lee Surn
Yes, compensation controls for flow cytometry should be prepared in the same experiment. Running compensation controls separately from your experiment can lead to inaccurate compensation and unreliable data.
The autofluorescence of your cells, especially in unstained controls, can change depending on their state (e.g., activation, treatment, or death). Running the unstained control from a different session may not accurately capture the background fluorescence of your current experimental cells.
The primary purpose of single-stained controls is to determine the degree of spectral overlap (spillover) between different fluorophores. This requires running the controls in the same instrument and under the same conditions as the experimental samples to accurately calculate the compensation matrix.
The best practice would be to prepare a single-stained sample for every fluorophore used in multicolor panel. Include an unstained cellular control or compensation beads for every cell type and treatment condition tested in your experiment. For complex multicolor panels, FMO controls (samples stained with all antibodies except one) are essential for identifying positive/negative populations and improving gating reliability.
Best,
Hi Lee Surn
I have to admit I would not consider myself an absolute expert in flow but I ran into the issue of low expressed markers and scarce samples before so I know the struggle.
Totally agree with what Malcolm Nobre explained.
In addition to that I was wondering if part of your challenge is that you are looking for a positive control? If so I belive you could use other cells (if you have them) that are well known to express your marker of interest just to confirm your gating so to say. Another option would be to stimulate some of your cells in a way that they would express more of said marker (so you will not need as many of them to verify your gate).
Another usefull thing in general is to carefully select your fluorophores according to the expected abundance of your markers. So for example if you know CD31 is very low, make sure to measure this target using a very bright fluorophore (e.g. PE, FITC etc.) and vice versa.
Also there are some flow people out there who prefer using beads to establish the compensation matrix and there are people who prefer using the actual sample. If you are using a flow cytometer in a facility or under the supervision of someone who has been using your specific machine a lot, it might be very helpful to discuss this with them as well since they will for example know which fluorophores work best / are brightest with said machine.
Hope this could help a little :)
many greetings and good luck !
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