Table 1.
Total Receptor surface distribution of DAF and αvβ3 at Vero-E6, and Tanoue B cells surfaces.
| Cell | DAF a | Total αvβ3 b |
|---|---|---|
| Vero E6 | 5.85 ± 1.37 × 103 i | 1.61 ± 0.38 × 106 |
| 2.54 ± 0.84 × 105 ii | ||
| 2.26 ± 0.28 × 105 iii | ||
| Tanoue B | 9.29 ± 2.18 × 104 i | None detected |
Median channel fluorescence (MCF) from flow cytometry histograms was used to determine the receptor expression using MESF calibration beads (see Experimental methods). The same secondary antibody was used for all measurements. IgG1 and IgG 2a K isotype controls were used as appropriate isotype controls. (a) Several primary antibodies were used because of concerns about poor cross-species reactivity between the commercially available anti anti-human DAF clone BRIC 216 and DAF expressed on green monkey (Vero E6) cells. The results from the different clones are listed: (i) clone BRIC 216 (Millipore) (ii) IA10 (iii) 2H6 ascites. (b) Anti-Integrin αvβ3 antibody, clone 23C6, was used as primary antibody. Secondary antibody was an Alexa488 tagged goat anti anti-mouse. Adherent cells (Vero E6) were detached with Accutase (Sigma, St. Louis, MO, USA), blocked with 10% human serum for 1 h, and incubated with primary antibodies at 4 °C on a rotator for 2 h. Secondary antibodies were incubated at 4 °C on a rotator for 1 h. The cells were washed once and then analyzed with an Accuri C6 flow cytometer. Non-specific binding by secondary antibodies was determined by staining cells in the absence of primary antibodies.