Does anyone work with fixed human whole blood samples in order to stain CD11b with ICRF44 clone (FITC) for flow cytometry?

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Carolina Garcia de Alba

Hi Liliya

I'm not sure if I understand your experiment, but if you are going to work with whole blood samples and stain for CD11b-FITC and after that you are going to fix your cells I think you are safe. Is not the clone the problem when you are working with fixed cells in flow cytometry, is more the fluorochrome, the tandem fluorochromes (APC-cy7, PE-Cy5 etc) dissociate when you fix them so if you need to fix is better to use simple fluorochromes as FITC. Anyway you need to measure the performance of your antibody comparing the results in samples without fixation and samples fixed and stored for the amount of time you are going to store them (always in the dark and 4C).

I hope this helps.

Liliya N Kirpotina

Thank you Carolina,

in my case I have to fix cells first and then to stain the whole blood samples for CD11b activation, as described in the following paper:

A novel flow cytometric assay of human whole blood neutrophil and

monocyte CD11b levels: Upregulation by chemokines is related to

receptor expression, comparison with neutrophil shape change, and

effects of a chemokine receptor (CXCR2) antagonist

Grant C. Nicholsona, Rachel C. Tennanta, Donald C. Carpenterb, Henry M. Saraub,

Onn Min Konc, Peter J. Barnesd, Michael Salmonb, Rupert S. Vesseyb,

Ruth Tal-Singerb, Trevor T. Hansela,

I have CD11b-FITC, the simple fluorochrom, ICRF44 clone from eBiosciences. I was told by technical support, that this clone is not compatible with fixation. But I found the opposite results on BD website.

I would suggest then that you try to analyze the same sample fixed and without fixation and check for the percentage of positive cells and fluorescence intensity maybe of CD11b, if you obtain the same results then you are good to go!

Also, eBiosciences have their on fixation solution and (of course) they strongly suggest that you use that one because their results are validated with that product. So maybe is worth to try that too just to be safe.