Anti cd19 6d5

Cells were isolated 72 hours after injection with LNPs unless otherwise noted. Mice were perfused with 20 mL of 1X PBS through the right atrium. Liver and lung tissues were finely minced, and then placed in a digestive enzyme solution with Collagenase Type I (Sigma Aldrich), Collagenase XI (Sigma Aldrich) and Hyaluronidase (Sigma Aldrich) at 37 ºC at 550 rpm for 45 minutes^4,5. The spleen, bone marrow and thymus tissues were placed in 1X PBS solution. Cell suspension was filtered through 70μm mesh and red blood cells were lysed. Cells were stained to identify specific cell populations and sorted using the BD FacsFusion and BD Facs Aria IIIu cell sorters in the Georgia Institute of Technology Cellular Analysis Core. The antibody clones used were the following: anti‐CD31 (390, BioLegend), anti‐CD45.2 (104, BioLegend), anti‐CD68 (FA11, Biolegend), anti‐CD3 (17A2, Biolegend), anti‐CD19 (6D5, Biolegend), anti‐CD11b (M1/70, Biolegend), anit‐CD11c (N418, Biolegend), anti‐CD4 (GK1.5, Biolegend), anti‐CD8a (53‐6.7, Biolegend), anti‐CD34 (SA376A4, Biolegend), anti‐CD25 (3C7, Biolegend), anti‐CD326 (G8.8, Biolegend), and PE anti‐mCD47 (miap301, BioLegend). Representative flow gates are located in Supplementary Figure 3. PBS‐injected Ai14 mice were used to gate tdTomato populations for intravenous administration.

Single cell suspensions were generated from murine tissues of C57/BL6 and BALB/c mice. Afterwards, cells were treated with FcR-blocking reagent (Miltenyi Biotec) for 10 min at 4°C after washing with flow cytometry buffer. To exclude dead cells, samples were washed twice with PBS and incubated with BD Horizon™ Fixable Viability Stain 510 viability dye (for 10 min at RT in the dark). Subsequently surface staining to discriminate immune cell populations with PerCP anti-CD45 (30-F11, BD Biosciences), APC-Cy 7 anti-TCRβ (H57-597, BioLegend), Pe-Cy7 anti-CD11b (M1/70, BD Biosciences), and anti-CD19 (6D5, BioLegend), was performed. The data was acquired using a BD FACS LSR II instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc, version 9.9.6 or 10.4.1).