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 antige...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2002-03-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/02323dd05 |
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| Summary: | 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. |
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| ISSN: | 0736-6205 1940-9818 |