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Four color immunofluorescence detection using two 488-nm lasers on a Becton Dickinson FACS vantage flow cytometer

Four color immunofluorescence detection using two 488-nm lasers on a Becton Dickinson FACS vantage flow cytometer

Cytometry. 29(2): 178-181

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.

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Accession: 008701634

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PMID: 9332825

DOI: 10.1002/(sici)1097-0320(19971001)29:2<178::aid-cyto11>;2-t

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