Four color immunofluorescence detection using two 488-nm lasers on a Becton Dickinson FACS vantage flow cytometer
✍ Scribed by MaryAnn DeMaria; R. Paul Johnson; Michael Rosenzweig
- Book ID
- 101242170
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 100 KB
- Volume
- 29
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
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.