I am analyzing my cells of interest, such as B cells, when i apply gates from one sample to another, am facing the applied gate in the second sample seemed that it is missing some populations. Such as, in sample 1 the CD27+ CD21+ has 10,000 the level of expression and second sample the maximum expression is about 8000, thus when i apply the first gate to the second, i think i would miss some dim population in the second sample. FYI, the voltage for these sample is similar during acquisition. Further, am also seeing this even with sample which were stained together at the same time. Therefore, should leave the second sample as it is or move to include all CD27+CD21+ cells?
I will look forward for your assistance
Thank you very Much!!!!
Hi,
Different individuals can have different expression and "shape" to the populations.
You must adjust gates between different samples, and your control needs to be cells from the same sample/individual, but without the marker in question (FMO).
Kerstin
Apart from trying to use similar voltages and staining controls, I use one "simple" strategy in FlowJo: once I have a well-defined gating strategy, I create a template in flowjo (save as ==> template (WSTP)).
Then I apply that template to my new samples to analyze.
When you have the samples loaded in the workspace: 1. create the compensation matrix from your stain controls, 2. apply the matrix to the template. Now your samples will appear with the gating strategy previously created.
Now is time to double-check if the gates previously created do not differ substantially from your original gates.
I hope this helps a bit,
Juan C.
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