Four color immunofluorescence detection using two 488-nm lasers on a Becton Dickinson FACS Vantage flow cytometer.
Multiparameter flow cytometric analysis is sometimes limited by the availability of directly conjugated monoclonal antibodies or streptavidin-conjugated secondary reagents. While many manufacturers offer a wide variety of monoclonal antibodies or streptavidin reagents directly conjugated to 488-nm excitable fluorochromes, there are not many available that are directly conjugated to 360-nm (UV) or 630-nm (HeNe) excitable fluorochromes. For this reason we attempted to develop a four color immunofluorescence staining protocol on a FACS Vantage using four 488-nm excitable fluorochromes. The fixed configuration of the FACS Vantage limits the feasibility of using four 488-nm excitable fluorochrome simultaneously because of the five fluorescence detectors, two are always electronically delayed. This means that only three signals-FL1, FL2, and FL3-can be detected off the primary 488-nm laser beam. FL4 and FL5 are always delayed, only detecting signals off the second laser beam. Our instrument is configured with an ILT air cooled 488-nm laser in the second position that is used in order to conserve the Coherent Enterprise laser when using only 488-nm excitable fluorochromes. Because of this, we were able to develop a four-color immunofluorescence staining protocol using only 488-nm excitable fluorochromes.
DeMaria
M
M
Division of Immunology, New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772, USA.
Johnson
R P
RP
Rosenzweig
M
M
D015703:Antigens, CD / Q000097:blood; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000295:instrumentation; D005456:Fluorescent Dyes / Q000032:analysis; D006098:Granulocytes / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D007834:Lasers; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D009000:Monocytes / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D010641:Phenotype
D015703:Antigens, CD; D005456:Fluorescent Dyes