Cd45 bv711

Tumors were harvested and resuspended into single-cell solution in PBS as described above. Murine spleens were mashed through a 70 μm filter into a ACK lysing buffer (Gibco, #A1049201) and incubated for 3 minutes. A total of 10 mL of PBS was added after the RBC lysis and the suspension was centrifuged at 1,500 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in PBS. After Fc blocking (BioLegend, 101319; 1:100) and Live/Dead staining with zombie NIR (BioLegend, #423105; 1:1,000) was performed during a 10-minute incubation at 4°C, the samples were washed with PBS. Conjugated antibodies including CD45-BV711 (BioLegend, #103147; 1:200), CD4-PE (BioLegend, #100407; 1:100), CD8-Pe-Cy7 (BioLegend, #100721; 1:100), NK1.1-FITC (BioLegend, #108705; 1:100), B220-Pe-Cy5.5 (BioLegend, #103209; 1:100), CD11b-APC (BioLegend, #101211; 1:200), F4/80-BV785 (BioLegend, #123141; 1:200), GR1-Pe Tx red (BD Biosciences, # 562710; 1:200) were incubated with each sample at 4°C in the dark for 20 minutes and washed with FACS buffer (2% FBS in PBS). The samples were analyzed using BD LSRFortessa X-20 flow cytometer. Collected flow cytometry data were analyzed using FlowJo software.

Heart tissue was minced and enzymatically digested using collagenase I (450 U ml⁻¹), collagenase XI (60 U ml⁻¹), DNAse and hyaluronidase (60 U ml⁻¹) (Sigma-Aldrich). Single-cell suspensions were stained with CD45-PerCP/Cy5.5 (clone 30-F11, 1:600, 103132, BioLegend), CD64-APC (clone X54–5/7.1, 1:600, 139305, BioLegend), Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), Ly6C-FITC (clone HK1.4, 1:600, 128006, BioLegend), CD11b-BV510 (clone M1/70, 1:600, 101245, BioLegend) and DAPI (0.1%, F10347, Thermo Fisher Scientific). Blood samples from Ly6G^Tdtomato mice were stained with Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), CD115-BV605 (clone AFS98, 1:600, 135517, BioLegend), CD11b-APC (clone M1/70, 1:600, 101212, BioLegend), CD45-BV711 (30-F11, 1:600, 103147, BioLegend), CD3-APC/Cy7 (clone 17A2, 1:200, 100221, BioLegend), CD19-APC/Cy7 (clone 6D5, 1:300, 115529, BioLegend), B220-APC/Cy7 (clone RA3–6B2, 1:300, 103224, BioLegend), Nk1.1-APC/Cy7 (clone PK136, 1:300, 108724, BioLegend) and DAPI. Data were recorded on an LSRII flow cytometer with FACSDiva 6.1 and analyzed with FlowJo 10 software (BD Biosciences). For qRT–PCR measurements, cells were flow sorted on a FACSAria II (BD Biosciences) into 350 μl of lysis buffer (RNeasy Plus Micro Kit, Qiagen).

Select groups of mice received a ‘long-term’ dosage of Poly-A. Mice received a daily tail vein injection of 2 × 10⁸ (~1 µm) Poly-A particles in 100 µL sterile phosphate-buffered saline (PBS). Control mice received either an equivalent amount of PBS (‘saline’) alone or ‘free aspirin’—salicylic acid. Mice were weighed and underwent health scoring daily before euthanasia on the fifth day. At the time of euthanasia, a cardiac puncture blood draw was administered, and the liver was harvested. Blood was immediately placed on ice, blocked with 1 µL FC-receptor block (BioLegend #101320) for 10 min. Samples were then stained with 0.1 µg/mL CD45-BV711 (Biolegend #103147), 0.25 µg/mL CD11b-FITC (Biolegend #101206), and 0.1 µg/mL Ly6G-APC (Biolegend #127614) and lyse/fixed (eBioscience) before using an Attune flow cytometer to analyze circulating leukocyte populations. The liver was added to RPMI media with collagenase and DNAse and dissociated using a gentleMACS Dissociator (Miltenyi Biotec). The dissociated liver was strained, centrifuged, and the supernatant was utilized in an aspartate aminotransferase (AST) activity assay (Sigma Aldrich).

Cells were stained with BD Biosciences (Oxford, U.K.) mAbs: CD45-PerCP-Cy5.5 (30-F11), CD45.2-V450 (104), CD4-BV650 (RM4-5), CD3-FITC (17A2), CD11b-eFluor450 (M1/70), CD19-BV711 (1D3), SiglecF (E50-2440), CD103-PE-CF594 (M290), Ly6G-BV650 (1A8); eBioscience (Loughborough, U.K.) mAbs: CD4-allophycocyanin-eFluor780 (RM4-5); Invitrogen (Dublin, Ireland) mAbs: KLRG1-PE-eFluor610 (2F1) and CD127-PerCP-ef710 (SB/199); and BioLegend (London, U.K.) mAbs: CD45-BV711 (clone: 30-F11), CD3-BV605 (17A2), CD11b-allophycocyanin-Cy7 (M1/70), CD11c-PE-Cy7 (N418), Ly6G-BV785 (1A8), Ly6C-BV606 (HK1.4), and SiglecF-allophycocyanin (S1700L). Before surface staining, Fc receptors were blocked using Fc-Block CD16/32 (BD Biosciences), and cells were incubated with LIVE/DEAD Fixable Aqua stain (Molecular Probes, Invitrogen) to isolate dead cells. For staining of transcription factors, cells were fixed and permeabilized using the Foxp3 staining buffer kit (Invitrogen) and stained with mAbs: GATA3-PE (TWAJ) and Foxp3-PE-Cy7 (FJK-16s). For the detection of YFP, along with intracellular transcription factors from Rora-YFP mice, after surface markers and viability stain, cells were prefixed with 2% paraformaldehyde followed by Foxp3 staining buffer kit. Cells were analyzed using a BD Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR), using appropriate controls.