Cd16 32 fc block clone 2.4g2

Totally, 1.5 million cells from tumors in mTmG mice were stained following the BD Cytofix/Cytoperm Fixation/Permeabilization microplate staining protocol (BD Biosciences), using the following antibodies: CD16/32 Fc Block (clone 2.4G2, BD Biosciences cat. 553142, 1:100), CD45 (clone 30-F11, BioLegend cat. 103127, 1:350), PD-L1 (clone 10F.9G2, BioLegend cat. 124313, 1:100), CD11b (clone M1/70, BioLegend cat. 101223, 1:100), CD11c (clone N418, BioLegend cat. 117323, 1:100). Cell lines were stained with PD-L1 (clone 10F.9G2, BioLegend cat. 124307, 1:100). Dead cells were excluded using Ghost Dye Violet 510 Dead Cell Stain Kit (Tonbo Biosciences) according to manufacturer protocol, and compensation was calibrated with single-color controls on Ultracomp eBeads (Invitrogen). Cells were quantified using a Gallios 561 cytometer (Beckman Coulter) and analyzed using Kaluza analysis software (Beckman Coulter) with fluorescence-minus-one controls to verify gating.

Survival indicated by forward scatter (FSC)/side scatter (SSC) as well as GFP or DS-RED expression of transduced cells was assessed by flow cytometry using FACSCalibur or LSRFortessa (BD Biosciences). For a more detailed analysis of viability and apoptosis induction, the Annexin V-PE (phycoerythrin) apoptosis detection kit (BD Biosciences) in combination with 7-AAD (BD Biosciences) was used. For cell surface staining of single-cell suspensions, the following anti-mouse antibodies were used: CD16/32 (Fc Block, clone 2.4G2, BD Biosciences) and CD19-APC (eBio103), B220-PerCPCy5.5 (RA3-6B2), IgM-APCe780 (II/41), CD43-PE (eBioR2/60), CD24-FITC (30-F1), and CD11b-PECy7 (M1/70) (eBioscience).