I am the beginner of the flow cytometry . When I analyze my data of healthy PBMC
I found out more than 60% of my cell is on the chart edges.What cause this phenomenon..and how to solve this..I stain for 9 colors
voltage is following
FSC 430
SSC 300
V500 395
APC 540
ALexa F 700--560
APC-cy7 463
PE-427
PE-TR 450
BV421--320
FITC--380
PE-cy7 -- 450
Hello Yunuo,
there are actually two possibilities I can think of why you can have cells (in perfect condition) piling up on the axis (assuming you are using digital data, like on a Canto or LSR).
First, the voltages on the PMT is too low (as pointed out by Ajay). You could increase the voltages the next time you measure to make the graph ‘prettier’, but if the voltages on the PMT fall within the linear range of sensitivity (as measured by your core facility regularly with beads) your data are valid even if the cells end up on the axis.
Second, due to compensation some cells end up having negative values. As the regular graph does not display negative values the cells are piled up on the axis. This could be due to overcompensation, but can happen also with correctly compensated data. There is nothing bad or wrong about this and is just a consequence of the mathematics involved in the compensation.
However, if you don’t see your cells, you cannot make any statements on them. I strongly recommend using always (!) a biexponential display (as it is called in DiVa and in FlowJo). That way you see all your cells (also the once with negative values) and can see if the compensation is correct.
So my suggestion would be to look at your data again with a biexponential display and see if this is now easier to interpret.
Good luck, Gerhard
Go back to previous paper similar to your work and read about the average number of isolated cells, and ensure you are making the proper gating because you may have already enough cells and the remaining 60% is not important (fragments of destructed cells).
You should put in mind that in the flow each event is represented by a dot. This include even the fragments of cells or any other particles. If Your isolated cells are lower than previous papers then try to handle your sample gently to decrease destructed cells. Perform your steps quickly and on ice as much as you can to decrease number of dead cells.
Hello dear Wang,
The sole reason for this is that your voltage settings are not correct.
You need to play with voltage only as varying the same can result in the orientation of cells in central region of chart and do try to set the voltage for FSC and SSC with unlabelled cells and then go with your labelled ones as former will automatically set the same for labelled cells.
Hope it helps!
Tks Kumar..I repeat the experiment again..and reset all the compensation ..Now it works ..thanks for everyone's reply!!
Hello Yunuo,
there are actually two possibilities I can think of why you can have cells (in perfect condition) piling up on the axis (assuming you are using digital data, like on a Canto or LSR).
First, the voltages on the PMT is too low (as pointed out by Ajay). You could increase the voltages the next time you measure to make the graph ‘prettier’, but if the voltages on the PMT fall within the linear range of sensitivity (as measured by your core facility regularly with beads) your data are valid even if the cells end up on the axis.
Second, due to compensation some cells end up having negative values. As the regular graph does not display negative values the cells are piled up on the axis. This could be due to overcompensation, but can happen also with correctly compensated data. There is nothing bad or wrong about this and is just a consequence of the mathematics involved in the compensation.
However, if you don’t see your cells, you cannot make any statements on them. I strongly recommend using always (!) a biexponential display (as it is called in DiVa and in FlowJo). That way you see all your cells (also the once with negative values) and can see if the compensation is correct.
So my suggestion would be to look at your data again with a biexponential display and see if this is now easier to interpret.
Good luck, Gerhard
Thank you Gerhard,the explanation is quite detailed and easy to catch up with !
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