5 replies to this topic

[euglena]

Hey FACS experts,

I am staining PBMC's for regular markers like CD4, CD8, CD11b and B220, CD45 etc

My markers are all from BD Pharmingen and I usually stain them all in 1:200

Question is, what staining conditions do you use? I was using 15 minutes at room temperature in the dark. Someone told me it's too much. Should I put them in the fridge?

[Katie Hulse]

euglena, on Jul 28 2009, 02:34 AM, said:

Hey FACS experts,

I am staining PBMC's for regular markers like CD4, CD8, CD11b and B220, CD45 etc

My markers are all from BD Pharmingen and I usually stain them all in 1:200

Question is, what staining conditions do you use? I was using 15 minutes at room temperature in the dark. Someone told me it's too much. Should I put them in the fridge?

I have always stained PBMCs for 15min at room temp, with no issues.

[miBunny]

I stain 15 minutes on ice. Keeping the cells cold will minimize cellular internalization of antibody-bound receptors. For some antibodies, longer incubation times are necessary. I used to keep all my cells covered with foil during the staining step. I stopped doing this about 4 years ago and I haven't noticed a difference. The lab doesn't really have a direct source of bright sunlight and the overhead lights are not strong enough kill the fluorescence.

[Stephan]

I usually do about 20min in the dark at room temp. Seems to work fine. Why change a winning formula?

[flowgirl]

I attend a flow course recently and the researcher that taught the immunophenotyping class said most of the binding happens on the first minute of incubation (not true for IC staining). I usually do 20 min on ice, just in case.

Also (and I confess I hadn't done that) he said we should always titrate new batches of antibody, that we would be amazed with the differences between the same antibody from different batches.

So I would say 15-20 minutes, RT or ice will do the job

[Doki]

in the refrigerator for 20-30 mins. that will be dark and 4degC.