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. Author manuscript; available in PMC: 2014 Apr 15.
Published in final edited form as: Biochem Pharmacol. 2013 Jan 31;85(8):1171–1181. doi: 10.1016/j.bcp.2013.01.021

Figure 4.

Figure 4

Saturation binding assays with MRS5218 using FCM following incubation for 1 h with the hA3AR expressed in CHO cells in the absence or presence of different inhibitors of receptor internalization. Fluorescent intensity values of small molecule-cell conjugates were measured using a FACSCalibur flow cytometer (BD, Franklin Lakes, NJ) with a 635 nm laser, FL4 channel and log mode and Cell Quest Pro software (BD, Franklin Lakes, NJ). MFIs were converted to MESF (Figures S5 and S6) values using QuickCal program v. 2.3 (Bangs Laboratories, Inc., Fishers, IN). Nonspecific binding was measured in the presence of 10 µM MRS1220. The apparent Kd values were determined to be 16.1±4.3 nM (without blocking internalization), 31.4±4.6 nM (0.4 M sucrose in the media) and 46.9±23.5 nM (4 °C incubation). Results are expressed as mean ± S.E. (n=3).