Transfusion of TRPC6-KO RBCs into WT mice. (A) Microscopic fluorescence image of RBCs from a TRPC6-KO mouse loaded with the Ca²⁺ dye Fluo-4 (5 μM, 1 hour, 37°C) and immobilized with Cell-Tak). Microscopic imaging¹² was performed while the cells were stimulated with LPA (5 μM, 5 minutes). (B) Microscopic fluorescence image from the same experiment described in panel A but with RBCs from a WT mouse (C57Bl6/N). The full image sequences, where the images in panels A and B were taken from are provided in the supplemental Material. Scale bar, 40 μM (A-B). (C) Two hundred microliters of RBCs from total TRPC6-KO mice (hematocrit of 50% in a 0.9% NaCl solution) were transfused into WT mice (C57Bl6/N) by retrobulbar injection under isoflurane narcosis. First, TRPC6-KO RBCs were labeled with the dye PKH26 (Sigma-Aldrich)⁴ (dark red symbols). Alternatively, the TRPC6-KO mouse was crossbred with a mouse expressing tdRFP,¹⁹ that is, having RBCs with an intracellular fluorescent protein (light red symbols). Before transfusion and up to 14 days after transfusion, blood was sampled at the facial vein (10 μL) from the transfused mice. The relative number of PKH26/tdRFP-positive cells is provided in supplemental Figure 3A. RBCs were video imaged as shown in panels A and B for 15 minutes. The maximum value of the background-corrected, cellular F/F_o signal of PKH26- or tdRFP-positive cells was analyzed.⁴ Each plotted data point is the median of at least 100 RBCs from 1 mouse (natural decrease in cell number over time after transfusion). (D) Replot of the data presented in panel C at selected time points and comparison to WT RBCs from the transfused mice. (E) Same experiments as in panel C with all data fitted together (red symbols) and similar experiments as in panel C but with WT mice splenectomized 1 week before transfusion (gray symbols). For splenectomy, mice were anesthetized by an injection of ketamine (75 mg/kg body weight) and xylazine (15 mg/kg body weight). After abdominal sectioning, the splenic vein and splenic arterywere ligated, and the spleen was removed. The relative number of tdRFP-positive cells under the different experimental conditions is provided in supplemental Figure 3B. (F) Replot of the data presented in panel E at selected time points and comparison to WT RBCs from the transfused mice. In panels D and F, values are plotted as the mean ± standard deviation. N, number of animals. In comparison, “ns” denotes not significant (P ≥ .5), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. Significance was tested with ordinary 1-way analysis of variance with Tukey multiple comparison tests (Prism 9, GraphPad Software, San Diego, CA).