Ultra leaf purified anti mouse cd16 32

The co-cultured PBMC were stained with Fixable Viability Stain (Thermo, L34965) and Fc receptor blocking reagent [Ultra-LEAF™ Purified anti-mouse CD16/32 (101320, BioLegend)]. Next, they were stained with CD-3 (BD 557943), PD-1 (BD 561273), and CD8 antibody (thermo, A15448). The prepared single-cell suspensions were filtered through 40-μm nylon meshes (352340, Corning). Results were then acquired using BD Calibur, BD Fortessa, or Miltenyi MACSQuant systems. Data were analyzed with FlowJo_V10 software (TreeStar).

Chopped tumor tissues were enzymatically digested using the dissociation buffer supplemented with 1 mg/ml Collagenase B (11088807001, Roche) and 1 mg/ml Hyaluronidase (H3506, Sigma-Aldrich) for 1 h at 37 °C. The prepared single-cell suspensions were filtered through 40-μm nylon meshes (352340, Corning) and centrifuged at 400 g for 5 min. Then, the centrifuged cells were treated with red blood cell lysis buffer (C3702, Beyotime). Subsequently, the cells were dyed with Fixable Viability Stain 780 (565388, BD), and Fc receptors were blocked with Ultra-LEAF™ Purified anti-mouse CD16/32 (101320, BioLegend). Cells were then fluorescently stained with the following detection antibodies: α-CD45 (103132, BioLegend), α-CD3 (100206, BioLegend), α-CD8 (100706, BioLegend), α-Ki67 (151215, BioLegend), α-CD69 (104536, BioLegend), α-CD25 (102012, BioLegend), α-CD107a (121629, BioLegend), α-Granzyme-B (372214, BioLegend), α-IFN-γ (505838, BioLegend). Brilliant Stain Buffer (563794, BD Biosciences) and True-Nuclear™ Transcription Factor Buffer Set (424401, BioLegend) were used in this assay. Flow cytometry was performed using Beckman CytoFLEX LX, and the data were analyzed by FlowJo_V10.