Direct vs. Indirect Staining for Flow Cytometry

Flow cytometry is a high-throughput technique that analyzes cell populations at the single cell level. This assay relies on detecting fluorescence to identify specific intracellular and extracellular targets, which can be done through direct and indirect staining. Direct staining uses primary antibodies recognizing your target of interest that are conjugated to fluorophores. Indirect staining uses unlabeled primary antibodies that are subsequently detected by fluorophore-labeled secondary antibodies recognizing the species and isotype of the primary antibody. Both methods have advantages and limitations where understanding the details of your experiment will help you select the best protocol for your situation.