Cd45 2d1

Clear cell RCC patient tissue specimens were prepared by mechanical disruption using a razor blade followed by treatment with 280 U/mL Collagenase Type 3 (Worthington Biochemical) and 4 ug/mL DNase I (Sigma) at 37°C for 1 hour with periodic vortexing. Digested tissues were passed through 70 um filters. Resulting cells were resuspended in 44% Percoll – 66% Percoll gradient (Sigma) and centrifuged for 30 minutes at 1,900 g with no brake. Mononuclear cells were collected and immediately stained for flow cytometry analysis following Fc blocking (company) and live/dead staining (company). The antibodies used for flow cytometry were: HLA-DR (L243, Biolegend #307618), CD14 (HCD14; Biolegend #325608), CD45 (2D1; eBioscience 11–9459-42), CD16 (3G8; Biolegend 302008), CD56 (HCD56; Biolegend 318318), and CD3 (7D6; Invitrogen MHCD0317). Tumor-associated macrophages (TAM) were identified as CD45⁺CD3^–Lin^-HLA-DR⁺CD14⁺CD16⁺. Data are reported as the percent of all cells.

Human-specific antibodies were purchased from BD Biosciences (CD45, 2D1; CD3, SK7; CD4, RPA-T4; PD-1, MIH4), eBioscience (CD8, RPA-T8), BioLegend (PD-L1, 29E.2A3), R&D Systems (TIM-3, 344823), and Enzo Life Sciences (LAG-3, 17B4). Mouse-specific antibodies were purchased from BD Biosciences (CD3, 125-2C11; CD4, GK1.5; CD44, IM7; CD62L, MEL-14; PD-L1, MIH5; CD69, H1.2F3; IFN-γ, XMG1.2; TNF, MP6-XT22), eBioscience (CD8, 53-6.7; PD-1, J43; PD-L2, 122; Foxp3, FJK-16s; Ki67, SolA15; Granzyme B, NGZB), and BioLegend (CD45, 30-F11; PD-L1, 10F.9G2; CD25, PC61). Appropriate isotype controls were used where applicable. A viability dye was typically used to exclude dead cells. Intracellular staining was performed using the eBioscience Fixation and Permeabilization Buffer Kit. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) and ionomycin (750 ng/ml) for 4 h at 37°C, 5% CO₂ in the presence of 1 μg/ml brefeldin A (BD Biosciences). Surface staining was performed and cells were fixed and permeabilized with the BD Cytofix/Cytoperm Kit and stained for IFN-γ and TNF- α. Supernatant cytokines were measured by cytometric bead array according to the manufacturer’s instructions (Mouse Inflammation Kit; BD Biosciences). Data were acquired using a BD FACSAria or Fortessa LSR flow cytometer and analyzed using FlowJo software (Tree Star).