Washing during cell staining - FACS (Feb/04/2007 )

After incubation with antibody, the cells are washed with FACS buffer. How do you wash it? Some do it once and some do it twice, what difference does it make?

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After incubation with antibody, spin cells and get rid of supernatant.
Then wash with FACS buffer and vortex...then spin and get rid of supernatant.

The purpose of washing the cells is to get rid of unbound antibodies.
I normally wash cells twice...so no trace of unbound antibodies are left.

Hope this help.

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Thank you for the reply Minne.

What I have been doing is, after incubation, we fill up the tube with FACS buffer and spin down the cells. With that also in past we always got good results.

But, last time I did the filling up and spinning down step twice and though all the experimental conditions were the same, the results were significantly different than the ones I expected. I used different age of mouse too, so that might have caused the difference in result but wanted to know if washing twice can cause so much difference.

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