SURFACE ANTIGEN QUANTIFICATION
Quantitative Flow Cytometry (QCFM) can be used to analyze large amounts of cells for multiple antigens and for quantifying the number of binding sites per cell. Linear function represents the relationship between mean fluorescence intensity (MFI) of cells that were labeled with a fluorochrome that was attached to a ligand or monoclonal antibody (mAb) and mean number of receptors/cells or mean number of mAb binding sites/cell. Special calibrated beads are used to generate a standard curve that converts MFI into molecular equivalents of soluble fluorochrome (MESF) by binding a quantifiable amount of antibody (Antibody Binding Capacity – ABC). The example below shows quantification of anti-Human EGFR on the cell surface of Human squamous carcinoma cell line SQCCY1 with DAKO QIFFIT® – picture obtained from U.T.M.D. Anderson Cancer Center Science Park – Research Division website. Different populations of beads showed in the picture bind calibrated amounts of CD5-FITC antibody. This kit measures immunofluorescence indirectly.
You can use series of five fluorescent microsphere populations labeled with varying amounts of FITC or other known fluorophores like Alexa Fluor® 488, Alexa Fluor® 647, APC, Cy™5, PE-Cy™5, R-PE to measure expression levels of cells. Fluorescence intensity is quantified through direct comparison of fluorescence measurements from pure fluorochromes with those from microspheres surface-labeled with the same fluorochrome.
FREE analysis program (QuickCal® v.2.3) is provided with each kit to help you determine expression levels of cells, and also to evaluate instrument linearity and detection threshold. The analysis program can be obtained HERE and used by entering the Access Number that was provided with your standards.
Links And Useful Information
- Article on comparison of the three different techniques for direct and indirect immunofluorescence.
- Bangs Laboratories beads for MESF analysis
- How to use FCS Express to create channel calibration on your own data (video)
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.