Cd16 cd32 antibody 2.4g2

Samples of tumor, spleen, lymph node, and blood were harvested after killing the mice. Single-cell suspensions of tumors were obtained using a Tumor Dissociation Kit (Miltenyi). The single cells were then incubated in MACS buffer (PBS supplemented with 2% FBS and 1 mM ethylenediaminetetraacetic acid [EDTA]) containing 10 µg/mL CD16/CD32 antibody (2.4G2, BD PharMingen) for 30 min at 4 °C and then stained with the antibodies. Staining reagents included anti-CD45 (30-F11, BioLegend), anti-CD45 (30-F11, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD4 (RM4-5, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD25 (PC61, BioLegend), anti-CD44 (IM7, BioLegend), and anti-CD62L (MEL-14, BioLegend). Data were collected on an BD FACSAria Fusion flow cytometer and analyzed with FlowJo software (TreeStar). Dead cells and cell aggregates were excluded from analyses based on a LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (eBioscience), or DAPI, with forward scatter/side scatter characteristics. The immune cell gating strategy was as follows: T cell: CD45⁺ CD3⁺, CD4⁺ T cell: CD45⁺ CD3⁺ CD4⁺, CD8⁺ T cell: CD45⁺ CD3⁺ CD8⁺, B cell: CD45⁺ CD3⁻ CD19⁺.

Mice were euthanized at week 2 to 3 posttransplantation and tumors were harvested. Single-cell suspensions of mammary glands or tumors were prepared as described previously (80 (link)). Briefly, tissues were picked, minced, and further digested by 5 mg/mL Collagenase Type III (LS004182, Worthington), 0.001% (W/V) DNase 1 (D-4527, Sigma), and 1% (wt/wt) Dispase (17105041, Invitrogen) at 37 °C for 1 h. Cells were filtered with a 70-μm strainer before erythrocyte lysis with the lysis buffer (555899, BD Pharmingen). FcR was blocked by a CD16/CD32 antibody (2.4G2, BD Life Sciences) at the concentration of 0.5 mg per million cells before antibody staining. Antibodies were diluted for staining according to the manufacturer’s instructions. For CD206 intracellular staining, cells were fixed with IC Fixation Buffer (008222, eBioscience), permeabilized with Permeabilization Buffer (008333, eBioscence) and subsequently incubated with antibodies as described previously. Flow cytometry was performed with Gallious (Beckman) fluorescence activated cell sorting system and quantified by the FlowJo V10 software.