Quantification of red blood cell size and shape in AQP1−/−, RhAG−/−, and double‐knockout mice
Authors: Moss Fraser J. , Zhao Pan , Woost Philip G. , Jacobberger James W , Boron Walter F 
Published: November 2023

We are investigating the permeability of the red blood cell (RBC) plasma membrane to various gases. Our laboratory was the first to establish that the water channel aquaporin1 (AQP1) can also mediate CO2 transport. Other research groups later showed that AQPs can conduct NH3 and that rhesus (Rh) proteins also conduct CO2 and NH3. Collectively AQP1 and RhAG account for ~90% of the CO2 permeability of human RBCs. In other work presented at this meeting we are investigating the molecular basis for O2 transport (“Oxygen Channels in Red Blood Cells”, Pan Zhao et al.). That study employs blood from wild‐type (WT), AQP1−/−, RhAG−/−, and AQP1−/−/RhAG−/− double knockout (dKO) mice. In order to compare rate constants for the deoxygenation of hemoglobin during O2 efflux from RBCs (kO2) of each genotype generated in stopped flow (SF) experiments, and to corroborate the physiological data with mathematical modelling, we also need to determine if the RBCs from AQP1−/−, RhAG−/−, and dKO mice exhibit significant variations in size or shape compared to RBCs from WT mice. Blood from each genotype is processed according to the same protocol as for SF experiments and stained with calcein violet (CV; viability), thiazole orange (TO; to stain RNA), and DRAQ5 (to stain DNA). Nucleated blood cells (i.e. erythrocyte precursors and white blood cells) stain positive for CV, TO and DRAQ5; reticulocytes stain positive for CV and TO; and mature RBCs stain only for CV. Flow‐cytometry determines that both the forward‐scatter (cell size) and side‐scatter (granularity and projections) distributions are very similar among WT, AQP1−/−, RhAG−/−, and dKO RBCs taken from four age‐matched mice of each genotype, and that any sample to sample variations are due flow nuances. We also quantified the mean maximal diameter of RBCs from the same mice using an Imagestream flow cytometer. This device collects both brightfield and fluorescent images of individual cells during flow. Parameters can be analyzed as 2D spatial grids with intensity in the third dimension. We note no substantial difference in maximal RBC diameter among WT (6.8 ± 0.9 mm), AQP1−/− (6.7 ± 0.9 mm), RhAG−/− (6.5 ± 0.9 mm), and dKO (6.5 ± 0.9 mm) samples. However, both RhAG−/− and dKO blood contain a significantly higher percentage of nucleated blood cells and reticulocytes compared to WT. Although the nucleated blood cells and reticulocytes typically have 5–12% larger diameters than RBCs, when included with the mature RBCs in the size analyses (as they would be the case in a SF experiment), they do not contribute to a significant increase in mean maximal blood cell diameter because they only represent 4% (WT) to 7% (RhAG−/−) of the total cells in the sample. We conclude that RBCs among WT and the various KO mice do not differ appreciably in either maximal diameter or shape.Support or Funding InformationResearch grant support to W.F.B. was from the Office of Naval Research Grant N00014‐11‐1‐0889, N00014‐14‐1‐0716, N00014‐15‐1‐2060, the NIH U01‐GM111251 and the Meyer/Scarpa Chair.