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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| _version_ | 1850152739108028416 |
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| author | X.-P. Yang M. Gallo I. Ngan M. Nocerini M.M. Chen |
| author_facet | X.-P. Yang M. Gallo I. Ngan M. Nocerini M.M. Chen |
| author_sort | X.-P. Yang |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-930fc212e4674b19b80a91405f82b511 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2002-03-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-930fc212e4674b19b80a91405f82b5112025-08-20T02:25:54ZengTaylor & Francis GroupBioTechniques0736-62051940-98182002-03-0132367868210.2144/02323dd05Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody ScreeningX.-P. Yang0M. Gallo1I. Ngan2M. Nocerini3M.M. Chen41Abgenix, Fremont, CA, USA1Abgenix, Fremont, CA, USA1Abgenix, Fremont, CA, USA1Abgenix, Fremont, CA, USA1Abgenix, Fremont, CA, USACell-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.https://www.future-science.com/doi/10.2144/02323dd05 |
| spellingShingle | X.-P. Yang M. Gallo I. Ngan M. Nocerini M.M. Chen Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody Screening BioTechniques |
| title | Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody Screening |
| title_full | Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody Screening |
| title_fullStr | Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody Screening |
| title_full_unstemmed | Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody Screening |
| title_short | Use of CMFDA and CMTMR Fluorescent Dyes in FACS®-Based Antibody Screening |
| title_sort | use of cmfda and cmtmr fluorescent dyes in facs r based antibody screening |
| url | https://www.future-science.com/doi/10.2144/02323dd05 |
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