Use of CMFDA and CMTMR fluorescent dyes in FACS-based antibody screening.
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 thirdfluorochrome 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.
Yang
X P
XP
Abgenix, Fremont, CA 94555, USA.
Gallo
M
M
Ngan
I
I
Nocerini
M
M
Chen
M M
MM
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D010957:Plasmids; D012235:Rhodamines; D014162:Transfection
C116572:(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine; D000906:Antibodies; D000951:Antigens, Neoplasm; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012235:Rhodamines; C069306:5-chloromethylfluorescein