Is compensation done in flow cytometry can be applied to single sample or is it mandatory to apply the same compensation to all samples under study?

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Ravi Kumar Sharma

Hi Gaurav. Whenever you are having two dyes and if they show spillover, you simply have to compensate for every sample that you analyse. You just have to calculate compensation before your experiment and link the settings to your experiment, so that it gets linked to all your samples automatically. Compensation settings must be linked to experiment before acquisition, because they change with voltage also. So, the values at one set of voltages for fluorophores don't work for different set of voltages for same colours.

Good luck

Shen-An Hwang

You say you are using two dyes, but it's not that simple. You have to set compensation specific for the panel, meaning the specific antibody, and the dye that is linked to the antibody. So, if you use anti-CD3-FITC and anti-CD28-PE for panel A, but use anti-CD4-FITC and anti-CD8-PE for panel B, the compensation for panel A is not the same as panel B. Also, if your cell population you are analyzing are different, (such as splenocytes versus PBMCs) the compensation for each cell population will also be different. the antibody but also the cell population.

And yes, the compensation is part of the protocol setup for the machine before you run your samples, which means that compensation is applied to ALL samples run under that protocol. Even if you are trying "compensate" using an analysis program after the samples have been acquired. ALL samples run under that protocol will need to be analyzed using the same compensation.

It appears as if you might be new to flow cytometry. I would highly recommend you find someone in the lab where the flow cytometry is located to help you do the setup. it is very difficult to correct improper protocol setup AFTER you already have acquired the data.