Or are there any other advantages besides stopping/slowing any cell processes? If incubation over ice decreases non-specific binding, wouldn't it also decrease all antibody binding (also specific) in general? The cold is not selective...
Just a word of caution on antibody staining after fixation of cells. Some antibodies will not work on fixed cells. The crosslink disrupts the epitope in some manner. It is vital to check you still obtain the same population. As a pretty good rule of thumb if the same antibody clone is approved for IHC it should work. The addition of Fc-Block (anti-CD16/CD32) prior to Fixation or any other flow will greatly reduce no specific binding.
Best of luck
The fixation of the cells for flow cytometry avoids that the expression of surface or intracellular markers is modified, since the cell is killed and fixed in the way it is by crosslinking the different molecules/proteins etc. I think incubation in ice is done because slowing down the binding reaction, the antibodies have time to bind to the right antigen and not to the first random antigen that they find. So the answer is no, fixing the cells would not eliminate the need for incubation in ice, because fixation doesn´t have anything to do with the specificity of the antigen recognition by the antibody. Apart from this, you can also incubate at +4°C, but the specificity will decrease.
Just a word of caution on antibody staining after fixation of cells. Some antibodies will not work on fixed cells. The crosslink disrupts the epitope in some manner. It is vital to check you still obtain the same population. As a pretty good rule of thumb if the same antibody clone is approved for IHC it should work. The addition of Fc-Block (anti-CD16/CD32) prior to Fixation or any other flow will greatly reduce no specific binding.
Best of luck
Thank you to both, yes I optimised the method on a definite positive and eliminating the fixing step resulted in a significant decrease of a particular antibody. Since I will be fixing I wasn't sure of the need for incubation on ice. Thanks
We generally fix our cells after staining on ice or at room temperature. As others have noted fixing prior to staining can reduce or even eliminate the ability of your antibody to bind to the target. Staining on ice apparently can help prevent migration of targets in the cell membrane causing capping - this is what we were told in the past. Fixation after staining will as well. Fixing in paraformaldehyde (and processing samples in a BSC) helps reduce the potential for accidental exposure to infectious agents that may be present in clinical samples. We have processed samples for L&L analysis only to find out the patient had an infection rather than lymphoma.
John Powers earlier message about Fc block is important as well.
Ray
One should try both for comparison: staining after fixation on ice or at room temperature which mostly provides a faster binding of the antibody to the antigenic site.
Josef
No. even fixed cells must be kept in ice for all processing of staining for proper antibody-antigen interaction as well as reducing the stress on cell.
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