Cd8 53 6.72

Primary antibodies used for IHC and flow cytometry were as follows: c-Kit (2B8; eBioscience), CD16/32 (93; eBioscience), VE-cadherin (eBioscience), c-Kit (R&D Systems), Sca-1 (E13-161.7; BioLegend), CD48 (HM48-1; BioLegend), CD150 (TC15-12F12.2; BioLegend), IL-7Rα (SB/199; BioLegend), endoglin (MJ7/18; BioLegend), CD4 (L3T4; BD), CD8 (53-6.72; BD), B220 (RA3-6B2; BD), TER-119 (BD), Gr-1 (RB6-8C5; BD), CD34 (RAM34; BD), Mac-1 (M/70; BD), Flt-3 (A2F10.1; BD), CD41 (MWReg30; BD), CD45.2 (104; BD), CD45.1 (A20; BD), GPIbα (Xia.G5; Emfret), Clec2 (AbD Serotec), and Thpo (Bioss). Secondary antibodies for IHC were Alexa Fluor 488–conjugated IgGs (Molecular Probes) or Cy3/Cy5/DyLight549/DyLight649-conjugated IgGs (Jackson ImmunoResearch Laboratories, Inc.). IHC specimens were treated with DAPI (Molecular Probes) for nuclear staining. Stimulatory rabbit anti–mouse CLEC-2 antibody was a gift of K. Suzuki-Inoue.

Procedures of isolation and staining of erythroid populations and EBI macrophages were described previously (Chen et al., 2009 (link); Flygare et al., 2011 (link); Yang et al., 2021 (link)). The following antibodies were used for flow cytometric analysis: F4/80 (BM8; BioLegend), Vcam1 (MVCAM.A; BioLegend), CD169 (3D6.112; BioLegend), TER-119 (Ter119; BD Bioscience), CD44 (IM7; BD Bioscience), Sirpα (P84; BioLegend), CD71(C2; BD Bioscience), c-Kit (2B8; Biolegend), Sca-1 (D7; BioLegend), CD4 (RM4-5; BD Bioscience), CD8 (53-6.72; BD Bioscience), B220 (RA3-6B2; BD Bioscience), Gr-1 (RB6-8C5; BD Bioscience), and Mac-1 (M/70; BD Bioscience). For measuring ATP content and proliferation, early erythroblasts were sorted in 96-well plates and measured for luminescence via CellTiter-Glo 2.0 Assay (Promega). Flow cytometric cell sorting was performed using Aria II (BD Biosciences) and analyzed using FlowJo software package (Tree Star).