Fluorescence microscopic and flow cytometric analysis of the characteristics of red blood cells from wild-type and mutant mice.

(A-P) Red blood cell preparations from wild-type,apoE^−/−, SR-BI^−/−, andSR-BI^−/−/apoE^−/−mice were simultaneously stained with 2 lysosomal markers: Lysotracker (low-pH indicator, red) and FDG (lysosomal beta-galactosidase substrate, green), and were examined by phase contrast (panels A-D) and fluorescence (panels E-P) microscopy. Panels E-H show Lysotracker staining; panels I-L, FDG staining; and panels M-P, merged red and green fluorescence images. Yellow indicates spatial overlap of the signals. (Q-T) Dual-color fluorescence flow cytometric analysis of red blood cells from the indicated mice stained with thiazole orange to detect RNA (x-axis) and a phycoerythrin-conjugated monoclonal anti-Trfr antibody to detect cell-surface Trfr (y-axis). For each cell analyzed, the relative intensities of thiazole orange fluorescence and phycoerythrin fluorescence are indicated by a dot and presented on log scales. Each panel represents analysis of 10 000 cells. Dots in the lower left quadrant of the cytogram represent double-negative, mature erythrocytes.