Diva software 8

Blood samples were collected into D-PBS containing 10 mM EDTA via a small transverse cut in the tail vein. A 2% dextran (Pharmacosmos) was added to remove red blood cells. To further eliminate red blood cells, the remaining blood cells were treated with ammonium-chloride-potassium lysis buffer on ice for 5 min. After a 45–60-min antibody incubation on ice, samples were suspended in D-PBS with 2% FBS and 4,6-Diamidino-2-phenylindole to distinguish dead cells. Cells were analyzed and sorted using the FACS-Aria cell sorters. Antibodies were obtained from eBioscience (currently Life Technologies/Thermo Fisher) and BioLegend (Supplementary Table 2). Flow cytometry data were analyzed using Diva software 8.0.1 (BD Biosciences).

Blood collected from a small transverse cut in the tail vein was added to phosphate-buffered saline (PBS) supplemented with 10 mM EDTA and treated with 2% dextran at room temperature for 30 min to remove the red blood cells. The collected samples were lysed on ice using ACK lysis buffer (150 mM NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA) for 5 min to further remove the residue red blood cells. The samples were then incubated with mixed antibodies as described previously^{29 (link),32 ,34 (link)} for 30 min at 4 °C and finally resuspended in PBS supplemented with 2% FBS and DAPI. Antibodies were purchased from eBioscience (currently Life Technologies/Thermo Fisher) and BioLegend, as indicated in our previous reports. The cells were sorted using BD FACS-Aria I and II flow cytometers into granulocyte, B, CD4 T, and CD8 T cell fractions. The CD45 marker was analyzed to distinguish donor and recipient blood cells. Data obtained via flow cytometry were analyzed using Diva software 8.0.1 (BD Biosciences, San Jose, CA). The following cell surface markers were used to harvest 4 types of hematopoietic cell populations:
Granulocytes: CD4⁻/CD8⁻/B220⁻/CD19⁻/Mac1⁺/Gr1⁺/side scatter ^high;
B cells: CD4⁻/CD8⁻/Gr1⁻/Mac1⁻/B220⁺/CD19⁺;
CD4 T cells: B220⁻/CD19⁻/Mac1⁻/Gr1⁻/TCRab⁺/CD4⁺/CD8⁻; and
CD8 T cells: B220⁻/CD19⁻/Mac1⁻/Gr1⁻/TCRab⁺/CD4⁻/CD8⁺.