Anti ly6g apc cy7

Splenocytes isolated from control and TCL1 leukemia-bearing mice were suspended at a density of 4 × 10⁶ cells/ml, incubated with 1.0 µM CM-H₂-DCFDA fluorescent probe (Molecular Probes, Eugene, OR, USA) in PBS at 37 °C for 30 min and then washed with PBS. Next, for distinguishing the population of living granulocytes, the cells were incubated with a Zombie Aqua™ Fixable Viability Kit (BioLegend) for 20 min, washed with PBS and subsequently stained with the following fluorochrome-conjugated antibodies: anti-CD11b-PE (eBiosciences, San Diego, CA, USA), anti-Ly6C-PerCP-Cy5.5 and anti-Ly6G-APC-Cy7 (both from BD Bioscience). After a final wash with PBS, to determine the intracellular ROS levels, the geometric mean fluorescence intensity (MFI) of oxidized CM-H₂-DCFDA in CD11b⁺ Ly6G⁺ cells was analyzed by flow cytometry.

Splenocytes obtained from control or treated tumor bearing mice were liquid-nitrogen frozen and thawed before tests. Then, for lymphoid cell analysis, they were stained in a one-step test with the following fluorophore-labeled anti-mouse monoclonal antibodies (mAbs): anti-CD4-APC (BD Pharmingen, USA, RM4-5), anti-CD8-PE-Cy7 (BD Pharmingen, USA, 53–6.7), anti-CD49b-PE (BD Pharmingen, USA, DX5) and anti-CD19-FITC (BD Pharmingen, USA, 1D3). Phenotype analysis was carried out using the Becton Dickinson FACSCalibur apparatus with Cell Quest Software. For myeloid cell characteristics, the flow cytometry analysis of MDSC surface phenotype was performed as described previously [16 (link)] using fluorophore-labeled anti-mouse mAbs: anti-CD11b-PerCP-Cy5.5 (BD Pharmingen, USA, M1/70), anti-B220-APC (BD Pharmingen, USA, RA3-6B2), anti-Ly6G-APC-Cy7 (BD Pharmingen, USA, 1A8), anti-Ly6C-PE (BD Pharmingen, USA, AL-21) and anti-MHCII-FITC (BD Pharmingen, USA, 25-9-17). The cells were stained for 45 min at 4°C. The viability of spleen cells was assessed by incubation with DAPI dye. The analysis was carried out using Becton Dickinson FACSFortessa apparatus with FACSDiva software.

To analyze recruited inflammatory cells the air-pouches were rinsed with 3 mL of ice-cold PBS, softly kneaded, and exudates were collected. The liquids obtained were centrifuged (5 min, 350 ×g, 4°C). Staining with the Zombie Red Fixable Viability Kit (1:2000, BioLegend) was performed to exclude dead cells. After washing (3% FBS/PBS), cells were incubated (20 min, 4°C) with the following fluorophore-labeled antibodies (BioLegend, if not stated otherwise) to identify major leukocyte populations: anti-CD16/CD32 (1:100, clone 93), anti-CD45-APC-Cy7 (1:150, 30-F11), anti-CD11b-APC (1:800, M1/70), anti-CD11c-AF488 (1:400, N418), anti-Ly6G-APC-Cy7 (1:150, 1A8), anti-SiglecF-PerCP-Cy5.5 (1:100, E50-2440, BD Biosciences), anti-F4/80-PE-Cy7 (1:100, BM8), anti-I-A/I-E (MHC II)-BV421 (1:120, M5/114.15.2), anti-CD19-AF488 (1:150, 6D5), anti-CD3ε-APC (1:120, 145-2C11), anti-CD4-PerCP-Cy5.5 (1:200, RM4-4), anti-CD8a-PE-Cy7 (1:600, 53-6.7), anti-CD152 (CTLA-4)-PE (1:100, UC10-4B9). Fluorescence minus one control was used for CTLA-4, CD11c, and MHC II. After staining, the cells were fixed with 2% formaldehyde (10 min, 4°C), washed, and measured by FACSAria IIIu (BD). The data was analyzed in FlowJo v.10.7 (BD). For further information on the antibodies and their targets, see Table 1.