variable cell counts for staining

[brettsportler]

Dear all,

I am using FACS to evaluate receptor expression on PMN. For each experiment, I got different groups (they only differ in the volatile anesthetic they are incubated with, the rest of the procedure is standardised and remains the same for all PMN). I therefore assume that there should not be any significant changes in cell count between groups. Regarding the AB-staining, would changes in PMN-counts (say +/- 30%) be a problem for further inter-group analysis? PMN are washed after staining...

Thanks

[Warthaug]

I isolated neutrophils for years, and can tell you that a 30% difference in cell numbers post-isolation is pretty damned good. There can be huge variation in neut numbers between patients, and huge differences between isolations as well. 30% differences are something to be proud of.

As for your experiment, its hard to say what differences that 30% will do. One option would be to equalize everything; do a cell count on all your samples, and dilute everything so they all have the same concentration. If using any kind of biochemical output you'll need to do this, as most of those assays are highly sensitive to cell numbers/protein concentrations.

But it sounds like you're primarily staining and counting cells via FACS. If so, a 30% difference shouldn't make a huge difference; just make sure you're using an excess of antibody for your labeling and you should be fine.

Bryan

[brettsportler]

Dear Bryan,

I´m isolating my neuts and then aliquot equal amounts of PMN suspension for my different groups, so PMN numbers at the beginning of the experiment should be the same in each group (as I only compare the groups of one day=one neutrophil isolation, changes from day to day in absolute PMN numbers after isolation shouldn´t be a problem). Thing is that it is possible that, at the end of my experiments, there are differences in cell counts, although these differences shouldn´t be too big as all groups are handled the same way. But if, for example, I stain 1x10*6 PMN/ml with the same amount of mAB as 1,5x10*6 PMN/ml, would this change my FACS results?!