Flow cytometry can be performed directly using a fluorescently labeled primary antibody, or indirectly using an unconjugated primary antibody with a labeled secondary. Most researchers prefer direct detection due to its simplicity and ease of use. However, certain fluorescent antibody experiments may require indirect detection with a labeled secondary.

  1. The marker being detected is poorly expressed and signal amplification is required. An indirect detection method allows for multiple secondary antibodies to bind each primary antibody, thereby significantly amplifying the signal.
  2. The marker being detected is uncommon and a directly conjugated primary antibody is not available. For these cases, researchers can either perform their own conjugation using an antibody labeling kit or simply use an indirect detection method.
  3. The experiment calls for biotinylated antibodies detected using streptavidin conjugated to a fluorophore.


Choosing a Secondary Antibody

Indirect flow cytometry allows the choice of a wide range of fluorophores, enabling researchers to match their primary antibody with any desired label. Secondary antibodies can detect either whole molecule immunoglobulin or Ig fragments. For example, secondaries that are specific for the Fc region may be preferred because they do not interact with the primary antibody’s antigen-binding region. Fortunately, indirect staining methods can be combined with direct staining protocols and incorporated into multicolor staining experiments. FluoroFinder supports multicolor panel design with both direct and indirect detection methods. Simply select the primary/secondary option during marker selection to indicate which markers require secondary antibodies.    Additionally, you can match any unconjugated antibody in the FluoroFinder database to the fluorophore of your choice with the Match Pair Search Match Pair Search function. Simply search for your marker, select an antibody, and see available fluorophore options among matching secondary antibodies or conjugation kits and services.