Cd11b bv650

Dissociation of fresh tumor samples and antibody staining was performed as described previously^{40 (link)}. Cells were blocked with anti-mouse CD16/CD32 FC block (Biolegend, 1:100) for 10 min on ice and stained with Zombie Aqua Fixable Viability Kit (Biolegend, 1:500) to discriminate live and dead cells. The following antibody cocktails were used: CD4 BUV805 (BD, 1:100), CD3εBUV395 (BD, 1:20), CD8a BV785 (Biolegend, 1:100), CD25 BV650 (Biolegend, 1:50), TCRγ/δ BV421 (Biolegend, 1:100), CD62L PE (Biolegend, 1:500), CD44 APC-Fire (Biolegend, 1:30), CD45 PerCP Cy5.5 (Biolegend, 1:100), CD19 FITC (Biolegend, 1:100), EpCAM APC/AF647 (Biolegend, 1:200) for acquisition of adaptive immune cells; CD11c BUV737 (BD, 1:30), NK1.1 BUV395 (BD, 1:25), Ly6C BV785 (Biolegend, 1:200), CD11b BV650 (Biolegend, 1:100), F4/80 BV421/PB (Biolegend, 1:30), CD45 PerCP Cy5.5 (Biolegend, 1:100), Ly6G PE (Biolegend, 1:200), CD68 APC-CY7 (Biolegend, 1:20), EpCAM APC/AF647 (Biolegend, 1:200) for acquisition of innate immune cells. 1 × 10⁶  events were acquired per antibody panel on the BD LSRFortessa. Flow cytometry data were analyzed using FlowJo software (v10.6.2).

Peritoneal immune cells were harvested as described previously (98 (link)). Briefly, mice treated with LNPs containing Cy5-mRNA were euthanized before peritoneal cell harvest. Ice-cold PBS (5 ml; Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) was injected into the peritoneal cavity, which was gently massaged to dislodge attached cells into the solution. PBS was subsequently recollected into the syringe, ensuring minimal blood contamination, and transferred to tubes kept on ice. Cells were collected by centrifugation at 500g and resuspended in PBS and 5% FBS for staining. Cells were stained with the following antibodies in the presence of 1:1000 TruStain FcX antibody (BioLegend): CD19-BV421 (BioLegend, 115537), CD3–fluorescein isothiocyanate (BioLegend, 100203), CD11b-BV650 (BioLegend, 101239), and F4/80-allophycocyanin/Cy7 (BioLegend, 157315), all diluted 1:100. Flow cytometry was performed using a NovoCyte 3000 flow cytometer, and data were analyzed in NovoExpress (Agilent, Santa Clara, CA).

Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).

As before,
¹⁰ (link) 2 h before sacrifice, mice were intraperitoneally injected with methoxy‐X04 5 mg/kg (2 mg/ml dissolved 1 to 1 into dimethyl sulfoxide (DMSO) and resuspended 0.9% saline solution). After intracardial perfusion, microglia was isolated by mechanical dissociation and density gradient. The density gradient was generated resuspending the cell pellet in 70% Percoll in PBS overlayed with 37% Percoll solution in PBS and centrifuged at 2000 g for 20 min in a refrigerated centrifuge. Microglia was obtained by the 37‐70% Percoll gradient and washed 1:5 in ice‐cold PBS and spun at maximum speed in refrigerated microcentrifuge for 1 min. Microglia cells were isolated with FACSAria™ III cell sorter (BD Biosciences) after staining with CD11b‐BV650 (1:200 Biolegend, 141723), CD45‐BV786 (1:200, BD Biosciences 564225), and CX3CR1‐FITC (1:100, Biolegend, 149019). Microglia were gated as live (propidium iodide negative), CD11b+, CD45+, CX3CR1+ single cells, and Me‐X04⁺ and Me‐X04⁻ microglial populations in transgenic 5xFAD mice were sorted separately for further analysis (Supplementary Figure S2). The flow cytometry results were analyzed using FlowJo™ Software v10.8 Software (Becton, Dickinson and Company; 2023).