Anti mouse cd16 cd32 antibody

FCM analyses and cell sorting for blood³⁰ (link) and tissue⁶⁴ (link) cells were performed as previously described. lood samples were mixed with PBS, and red blood cells were lysed using the BD Pharm Lyse solution (BD Biosciences, Franklin Lakes, NJ, USA). Zombie Violet (BD Biosciences) treatment was performed to eliminate dead cells. The samples were then incubated on ice with an anti-mouse CD16/CD32 antibody (BD Biosciences, 1:100) to block Fc receptors. The cells were incubated with the fluorescence-labelled antibodies listed in key resources table on ice for 30 min. After centrifugation, the pellets were resuspended in 250 μl of PBS containing 2% fetal bovine serum. Flow cytometry was performed using a Gallios flow cytometer (Beckman Coulter). The data were analyzed using FlowJo software (version 7.6.5; Treestar, Ashland, OR, USA). For FCM analysis of brain and lung tissues, tissues were dissociated into single cells using their respective dissociation kit following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany), and the dissociated cells were processed as blood samples using the same method. Total RNA was collected from sorted cells and fixed overnight with a cell cover (Anacyte Laboratories, Hamburg, Germany).¹⁴ (link)

Single-cell suspensions from perfused lungs were prepared via enzymatic digestion using the Lung Dissociation Kit (Miltenyi) and following filtration through a 40 µm cell strainer. Red blood cells in the lung digestions and blood samples were removed using RBC lysis buffer (Biolegend). The samples were blocked with antimouse CD16/CD32 antibody (BD bioscience), and stained with DAPI and fluorescent antibodies to following antigens; CD45 (30-F11), F4/80 (BM8), CD11b (M1/70), Ly6C (HK1.4), CD115 (AFS98), Ly6G (1A8), CD11c (N418), CD14 (Sa14-2), CD36 (HM36), CD64 (X54-5/7.1), CD206 (C068C2), CCR5 (HM-CCR5), PD-L1 (10F.9G2), CD80 (16-10A1), CD86 (GL-1) all from Biolegend, and PD-L2 (122) from eBioscience. Flow cytometry was performed using LSRII cytometer (BD Biosciences) and analyzed using Flowjo software (TreeStar). In some experiments, the stained C-MOs (CD115⁺Ly6G⁻CD11b⁺Ly6C^high) in the blood or bone marrow, MAMPCs (F4/80⁺Ly6G⁻CD11b^highLy6C^high), MAMs (F4/80⁺Ly6G⁻CD11b^highLy6C^low), and RMACs (F4/80⁺Ly6G⁻CD11b^lowLy6C^low) in the lung of E0771-LG-injected mice were sorted using FACS Artia II (BD Biosciences).

Spleens, iliac and inguinal lymph nodes, and tail skin were harvested from CO₂ euthanized mice. To obtain single cell suspensions, spleens and lymph nodes were enzymatically digested with DNAse I (0.2 mg/ml; Roche) with and without Collagenase A (0.5 mg/ml; Roche), respectively, and processed as described previously [53] (link). Skin tissues were cut into fine pieces and incubated with collagenase IV (2 mg/ml; Worthington Biochemical Corp.) plus DNAse I (0.1 mg/ml) in RPMI 1640, supplemented with 10% fetal bovine serum and 1% penicilin/streptomycin, for 1 hr at 37°C. All cell suspensions were passed through 70 µM nylon mesh filters (BD Falcon) prior to lysis of erythrocytes with Ammonium-Chloride-Potassium Buffer (ACK; Lonza).
Blood was collected in heparinized tubes and erythrocytes removed by ACK lysis.
After blocking of Fc receptors by incubation with anti-mouse CD16/CD32 antibody (BD Pharmingen), surface-stainings were performed on single cell suspensions from tissues and blood using anti-mouse CD4-PerCP/Cy5.5 (clone RM 4-5; BD Pharmingen) and anti-mouse CD8a-FITC labeled antibodies (clone YTS; Abcam). Cells were fixed with Cytofix/Cytoperm (BD Biosciences) and acquisitions of flow cytometric data performed on a FACSCanto with FACSDiva software (BD Biosciences) [53] (link). The FlowJo software was used for analyses.

Whole de‐capsulated right kidneys were digested using Multi Tissue Dissociation Kit 2 on a gentleMacs Octa Dissociator (Miltenyi Biotec). Single‐cell suspensions were obtained by filtering the digested tissue through sterile 100 and 40 μm strainers. Red blood cells were lysed using ACK lysis buffer (Invitrogen; A1049201). Cells were re‐suspended in a 0.1% bovine serum albumin (BSA) solution and nonspecific Fc binding was blocked with an anti‐mouse CD16/CD32 antibody (BD Pharmingen) for 10 min on ice. After the blocking step, cells were incubated with fluorescent‐conjugated antibodies against CD45, F4/80, CD11c, CD19, Ly6G, CD3e, CD8a, and CD4 for 20 min on ice (Table 1). Data were acquired on a BD LSR Fortessa X‐20 flow cytometer using FACS DIVA software (BD Biosciences). Absolute number of CD45+ cells were normalized the total tissue mass and represented as CD45+ population/gram of tissue, while other cells were represented as percentage of CD45+ cells.

Splenocytes and TILs were analyzed by flow cytometry. Lymphocytes from tumor and spleens were isolated as previously described [31 (link),32 (link)]. Flow cytometry was performed as shown previously and anti-mouse CD16/CD32 antibody (BD Pharmingen) was used to block nonspecific binding [33 (link)]. The following fluorochrome-conjugated antibodies were used in 2×10⁶ cells: 0.4 μg CD45 (eBioscience, clone #30-F11), 0.4 μg CD3 (BD Pharmingen, clone #145-2C11), 0.4 μg CD4 (BioLegend, clone #GK1.5), 0.4 μg CD8 (eBioscience, clone #53-6.7), 0.4 μg CD11b (eBioscience clone #M1/70), 0.4 μg GR-1 (BD Pharmingen clone #RB6-8C5), and 1 μg Foxp3-Alexa488 (eBioscience, clone # FJK.16 s). Stained cells were acquired with FACS Canto flow cytometer (BD Bioscience) and 1×10⁶ to 2×10⁶ cells were analyzed with FlowJo software (Tree Star Inc.). Results are reported as total percentage of a cell population or ratio of cell quantities, as indicated.