Cd3e pe

Mice were bled into 150 µL of Alsever’s solution. Samples were then treated with 10 mL ACK buffer for 2 min and centrifuged at 1600 RPM for 5 min. The supernatant was removed, and pellets were washed in 5 mL sample media (SM, PBS with 2 mM EDTA and 2% FCS). Antibodies of Biolegend (San Diego, CA, USA): CD45.1 APC, CD45.2 pacific blue, Mac1 PE-Cy7, B220 APC-Cy7, CD3e PE, Lineage-PacificBlue (Including- anti Ter119, Mac1, Gr1, CD3e, CD4, CD8 and B220), c-Kit Alexa780, Sca1-APC, CD150-PEcy7, CD48- Percp cy5.5, CD9-APC, CD34-PE, CD84-PE. Cells were stained on ice for 1 hr and washed. Flow cytometry analysis of the reporter expression frequencies (ZsGreen+) and the surface markers’ expression were performed on the Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA). Fluorescence activated cell sorting (FACS) data analysis was performed using Kaluza v1.2 (Beckman Coulter). Sorting was performed on FACSAria III (BD), as previously reported [9 (link)].

Mice were tail bled into a 150-µl Alsever’s solution. Samples where then treated with 10 ml Ammonium-Chloride-Potassium (ACK) Lysing Buffer for 2 min and centrifuged (1600 rpm) for 5 min. The supernatant was removed, washed in 5 ml SM, centrifuged again, and finally plated in a 96-U well for 1–3 h for staining. Blood panel staining: CD45.1-Apc, CD45.2-PacBlue, Mac1-PeCy7, B220-ApcCy7, and CD3e-PE (all from Biolegend, San Diego, CA).

Antibodies used: CD45.1-Apc, CD45.2- PacBlue, Ter119-PerCPCy5.5, Mac1-PeCy7, B220-ApcCy7, CD3e-PE, cKit-ApcCy7, Sca1-APC, CD150-PeCy7, CD48--PerCPCy5.5, Gr1-PacBlue, and CD19-APC (all from Biolegend, San Diego, CA). Cells were stained on ice for 30 min, washed, filtered through a 70-µm filter, and analyzed using Fluorescence-Activated Cell Sort machine (FACS). Routine analysis was performed by using 4-laser Gallios (Beckman Coulter) and the Kaluza analysis software (Version 2.1). Sorting used of a 6-laser, BD FACSAria™ III sorter.