Confirmation of the SSEA1 surface antigen on immobilized stem cells with immunofluorescence microscope
To verify the results obtained by the OI-RD microscope, we monitored the change in SSEA1 surface antigens by conventional fluorescence microscopy. Right after printing, BSA spots and cells can be seen on the glass slide under the conventional (bright-field) microscope (Figure 4, top panels). The glass slide was incubated with the anti-SSEA1 antibody overnight, washed with PBS three times, and then incubated with FITC-conjugated secondary antibody. After washing, extraneous material (such as salt crystals from the printing buffer) were removed, and only the immobilized cells were visible under the conventional (bright field) microscope (Figure 4, middle panels). Under the fluorescent microscope, we observed that the anti-SSEA1 antibody indeed bound to pluripotent or differentiated stem cells, but not HEK293T cells or A19 fibroblast cells (Figure 4, bottom panels). These results confirm the findings with the OI-RD microscope. However, the OI-RD measurement provides additional information on the binding reaction kinetics that is lacking from fluorescence-based detection.
Figure 4. Immunofluorescence detection of cells with the stage-specific embryonic antigen 1 (SSEA1) marker. (Click to enlarge)
In conclusion, we show that the OI-RD microscopy method can be used to (i) detect the association between unlabeled antibody and a surface antigen; (ii) measure the binding affinities (e.g., equilibrium dissociation constant or Kd) between antibody and antigen; and (iii) detect hundreds of samples on a microarray platform. This report is the first study to directly measure the binding affinity between antibody and antigen on cell surface. In contrast to traditional cell surface studies, in which only the relative number of cell surface antigens can be determined, OI-RD microscope enables a dynamic characterization of surface antigens. With this powerful tool, we can detect the amount of and changes in affinity of cell surface markers as an independent monitor of developmental stages. Furthermore, this technique may be more generally used to detect the change in cell surface markers on many other cell types for research and clinical applications.
Acknowledgments
We thank Zhao-Qi Wang (Leibniz Institute for Age Research) for the A19 fibroblasts and U-Ging Lo for reagent preparation. This work was supported by grants from the National Institutes of Health (NIH; grants nos. R01NS061983 and R01ES015988 to W.D., and R01-HG3827 to X.D.Z.), the National Multiple Sclerosis Society (to W.D.), and Shriners Hospitals for Children (to W.D.). This paper is subject to the NIH Public Access Policy.
Competing interests
The authors declare no competing interests.
Correspondence
Address correspondence to Wenbin Deng (stem cells), Department of Cell Biology and Human Anatomy, Institute of Pediatric Regenerative Medicine, School of Medicine, University of California–Davis, Davis, CA, 95616, USA. e-mail: [email protected]; or Xiangdong Zhu (OI-RD microscope), Department of Physics, University of California–Davis, Davis, CA, 95616, USA. e-mail: [email protected]
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