Anti b220 apc cy7

The following antibodies were purchased from Biolegend Inc.: anti-CD3-pacific blue (17A2), anti-CD4-pacific blue (GK1.5), anti-CD4-FITC (GK1.5), anti-CD8-pacific blue (53-6.7), anti-CD8-APC (53-6.7), anti-NK1.1-pacific blue (PK136), anti-CD11b-pacific blue (M1/70), anti-CD11c-pacific blue (N418), anti-CD11c-percp-cy5.5 (N418), anti-I-Ab-FITC (KH74), anti-I-Ab-PE (KH74), anti-CD34-PE (MEC14.7), anti-B220-APC-Cy7 (RA3-61B2), anti-B220-pacific blue (RA3-61B2), anti-CD117-PE-Cy7 (2B8), anti-PDCA1-APC (927), anti-CD135-APC (A2F10), anti-CD172-biotin (P84), anti-IL-17a-PE (TC11-18H10.1), and strepedavidin-PE-Cy7 (Biolegend, CA). The anti-WNT1 antibody was purchased from affinity Bioscience (Affinity, OH).

The immune surface phenotype was assessed for myeloid, lymphoid, mast cell, and multipotent HSPC marker: Anti-CD3 $\epsilon$ /APC-Cy7 (BioLegend Catalogue no.100330), anti-B220-APC/Cy7 (BioLegend Catalogue No. 103224), anti-Ter119/APC-Cy7 (BioLegend Catalogue No. 103224), anti-CD11b/AF700 (BioLegend Catalogue No. 103224), anti-Gr1 (BioLegend Catalogue No. 108422), anti-Fc $\epsilon$ R1 $\alpha$ /FITC (eBioscience Catalogue No. 11-5898-85) or anti-Fc $\epsilon$ R1 $\alpha$ /PE (eBioscience, Catalogue No. 12-5898-83), anti-cKit/APC-Cy7 (BioLegend, Catalogue No. 105826), and anti-Sca-1/PerCP-Cy5.5 (eBioscience, Catalogue No. 45-5981-82). Samples were finally stained within 0.2 $\mathsf{\mu }$ g/mL DAPI (Sigma-Aldrich, Catalogue No. D9542) for dead cell exclusion. Flow cytometry was performed with the Cytoflex S (Beckman Coulter, Brea, CA, USA) cytometer, and data were analyzed with the CytExpert (Beckman Coulter) and FlowJo, Version 10.2 (Tree Star, Ashland, OR, USA) software.

The following conjugated anti-murine antibodies were used for flow cytometry: FITC-anti-CD45.1 (A20, #11-0453), FITC-anti-CD9 (MZ3, #11-0091), PE-Cy7-anti-CD45.2 (104, #12-0454), PE-anti-CD127 (A7R34, #12–1,271), efluor450-anti-CD19 (eBio1D3, #48-0193), efluor-anti-CD45.2 (104, #48-0454), PECy7-anti-CD117 (2B8, #25–1,171), efluor-anti-CD34 (RAM34, #50-0341; eBioscience); APC-Cy7–anti-B220 (RA3-6B2, #103224), APC-anti-CD22 (OX-97, #126110), PE-Cy7-anti-CD93 (AA4.1, 136506; Biolegend); PE-anti-CD22 (Cy34.1, #553384), APC-anti-CD11b (M1/70, #553312), PE-anti-BP1 (BP-1, #553735), FITC-anti-GR1 (RB6-8C5, #553127; BD Biosciences). CAR detection was performed using protein-L with PE-Streptavidin (BD Biosciences). The following conjugated anti-human antibodies were used: efluor45-anti-CD19 (HIB19, #48-0199), FITC-anti-CD19 (HIB19, #11-0199)), efluor-anti-CD34 (4H11, #48-0349), PE-Cy7-anti-CD11b (ICRF44, #25-0118), FITC-anti-CD14 (61D3, #11-0149), APC-anti-CD117 (104D2, #17–1,178), PE-anti-CD33 (WM-53, #12-0338), PerCP-Cy5.5-anti-CD45 (HI30, #45-0459; eBioscience); PE/Cy7-anti-CD10 (H110a, #312214, Biolegend); PE-anti-CD22 (S-HCL1, #347577) and PE-anti-HLA-DR (G46-6 #556644; BD Biosciences). Samples were analysed on a BD LSR-Fortessa (BD Biosciences), data collected using FACS Diva software and analysed using FLowJo version 9.6.4 (Treestar).

Anti-PNAd (MECA-79) mAb was purified using a size-exclusion column with size-exclusion resin (Toyopearl TSK HW55; Tosoh, Japan) from the ascites of mice inoculated with the hybridoma. Purified MECA-79 was labeled with the Alexa Fluor 488 Protein Labeling Kit (Thermo Fisher Scientific, Waltham, MA). APC anti-CD4, Pacific Blue anti-CD8, APC-Cy7 anti-B220, APC anti-gp38, and biotin anti-CD31 mAbs were purchased from Biolegend (San Diego, CA). Anti-L-selectin mAb (MEL-14) was generated from the hybridoma, and anti-integrin LFA-1 (αL) mAb (M17/4) was purchased from Bio X cell (Lebanon, NH). Streptavidin-BD Horizon V500 was obtained from BD Biosciences (San Jose, CA), and LPA (18:1) and blebbistatin were from Sigma-Aldrich (St. Louis, MO). Mouse CCL21 and mouse ICAM-1-Fc were from R&D Systems (Minneapolis, MN).

Single-cell suspensions from mouse spleens and were stained using the following fluorescence-labeled anti-mouse antibodies: from BD Biosciences, FITC anti-CD38 (BD558813; clone 90; dilution 1:500), BV421 anti-CD95/Fas (BD562633; clone Jo2; dilution 1/500), PE-Cy7 anti-CD86 (BD560582; clone GL1; dilution 1:400), PE anti-CD184/CXCR4 (BD561734; 2B11; dilution 1:250); from BioLegend, APC-Cy7 anti-B220 (103224; clone RA3-6B2; dilution 1:750) and AlexaFluor647 anti-pSer139-H2AX (613407; clone 2F3, dilution 1:200). For internal markers, cells were fixed and permeabilized with the BD Cytofix/Cytoperm fixation/permeabilization solution kit (BD Biosciences). Data were acquired on a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed using the FlowJo software package (BD Biosciences).