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General procedure for flow cytometry using a primary antibody and conjugated secondary antibody.
Download our membrane staining summary.
Indirect labeling requires two incubation steps, firstly with a primary antibody, then with a compatible secondary antibody. The secondary (and not the primary) antibody has the fluorescent dye (FITC, PE, Cy5®, etc) conjugated. Please note that this is a general protocol, and you may need to adapt it for your applications.
We recommend analysis on the same day. For extended storage (16 hr) as well as for greater flexibility in planning time on the cytometer, resuspend cells in 1% paraformaldehyde to prevent deterioration.
If you need to wait longer than 1 hr before analysis, you may need to fix the cells after step 5. This can preserve them for several days (this will stabilize the light scatter and inactivate most biohazardous agents). Controls will required fixation using the same procedure. Cells should not be fixed if they need to remain viable. There are several methods available. The fixation for different antigens will require optimization by the user.
N/B polystyrene/plastic tubes are not suitable for use with acetone
Add 1ml ice-cold acetone to each sample.
Mix gently. Place at -20°C for 5-10 min.
Centrifuge, wash twice in PBS 1% BSA.
Do not add sodium azide to buffers if you are concerned with recovering cell function, eg if cells are to be collected for functional assays. It inhibits metabolic activity.
Check out our free online flow cytometry training, designed to help you achieve the most from this powerful technique.