Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody Screening

XP Yang, M Gallo, I Ngan, M Nocerini, MM Chen - Biotechniques, 2002 - Future Science
XP Yang, M Gallo, I Ngan, M Nocerini, MM Chen
Biotechniques, 2002Future Science
Cell-based immunizations are often used when membrane antigens are difficult to purify. To
confirm that an antibody binding to the surface of a cell line is, in fact, binding to the desired
antigen, FACS® can be performed independently on two cell lines, a transfected cell line
expressing the antigen of interest and a control cell line not expressing the antigen.
Antibodies binding only to the transfected cell line are then selected for further analysis. This
approach can be challenging if a large number of antibodies need to be screened and the …
Cell-based immunizations are often used when membrane antigens are difficult to purify. To confirm that an antibody binding to the surface of a cell line is, in fact, binding to the desired antigen, FACS® can be performed independently on two cell lines, a transfected cell line expressing the antigen of interest and a control cell line not expressing the antigen. Antibodies binding only to the transfected cell line are then selected for further analysis. This approach can be challenging if a large number of antibodies need to be screened and the antibody quantities are limited. Here we describe a novel method that combines the above two steps of FACS screening into a single step, based on the use of two fluorochromes, CMFDA and CMTMR, to stain transfected and control cell lines, respectively. Antibodies conjugated to a third fluorochrome are then added to the combined cells, followed by three-color FACS analysis. The newly modified FACS method is simple, sensitive, and high throughput. It can be used for antibody screening in multiple cell lines simultaneously.
Future Science