Flow Cytometry Antibodies

Q: Why are there two or more cell populations observed in flow cytometry (FC)?

a. More than one cell population expressing target protein. You should check if there is adequate cell separation.

b. Cell doublets present. Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot. You should mix cells gently using a pipette before staining and before running on the cytometer.

Q: How should antibodies be selected in double-labeled immunofluorescence?

When performing double-labeling immunofluorescence experiments, the selection of appropriate antibodies is crucial for observing the co-localization of two antigenic proteins in cells. Below are the antibody selection options for different situations:

a. Two directly labeled antibodies: If available, two directly labeled primary antibodies with different fluorescent markers should be selected. This reduces the risk of cross-reactivity of the secondary antibodies and is easier to handle.

b. Two indirectly labeled antibodies: In this case, two primary antibodies with different host sources (Host), each paired with a different secondary antibody with different fluorescent markers, need to be selected to avoid cross-reactivity.

c. One direct-labeled antibody plus one indirectly labeled antibody: If one is from a direct-labeled primary antibody and the other is unlabeled, then a primary antibody of a different host source and the corresponding secondary antibody can be selected. Both antibodies can be incubated at the same time because the directly labeled primary antibody will not cross-react with the secondary antibody of the other primary antibody.

d. Two indirectly labeled antibodies of the same host origin: If the two primary antibodies are of the same host origin, it is necessary to incubate one primary antibody and use the corresponding secondary antibody for detection, then incubate the second primary antibody and use its corresponding secondary antibody. This avoids cross-reactivity between secondary antibodies of the same host source.