MerTK-mediated engulfment of pyrenocytes. (A) Cells of the erythroblastic islands prepared from wild-type or MerTK^−/− mice were dispersed in PBS/BSA/EDTA, and analyzed by FACS for MerTK expression. The MerTK-staining profile in SytoxBlue^low population (upper panel) with (blue) or without (red) primary antibody is shown. (B-C) Erythroblastic islands from wild-type and MerTK^−/− mice were cultured overnight in medium containing 1% methylcellulose, washed to remove erythroblasts, stained with PI, and analyzed on an FACSCanto II. The PI-staining profile in SytoxBlue^low population is shown. The experiments were performed 3 times, and the average percentage of cells containing extra PI-positive materials are shown with standard deviation (B). The PI-stained samples were observed by BioRevo fluorescence microscopy with objective lens’s magnification of 40 (C). (D) NIH3T3 MerTK transformants were incubated at 37°C for 2 hours with pHrodo-labeled pyrenocytes in serum-free medium with or without 1 μg/ml Protein S, and subjected to FACSAria II analysis. Experiments were performed three times, and the average percentages of pHrodo-positive cells are shown with standard deviation. (E). NIH3T3 and MerTK transformants were plated on Labo-Tek chamber cover glasses, incubated with pHrodo-labeled pyrenocytes in serum-free medium with or without protein S, and observed by BioRevo fluorescence microscopy with an objective lens magnification of ×40. Scale bar, 50 μm.