Pe conjugated anti mouse cd16 32

Mice bearing subcutaneous and orthotopic tumors were euthanized 7 days after IRE, and tumors were harvested and dissociated using a mouse tumor dissociation kit according to manufacturer’s recommendations (Miltenyi Biotec, Germany). For the analysis of tissue-derived lymphocytes, tumor tissues, LN, and spleens harvested from mice were chopped into small pieces and mashed through a 70 um strainer. The red blood cells were lysed by red cell lysis buffer (TIANGEN). Then, cells were washed with PBS containing 1% FBS and stained with FITC-conjugated anti-mouse CD206 (141705, Biolegend, San Diego, USA), PE-conjugated anti-mouse CD16/32 (101307, Biolegend), APC-conjugated anti-mouse F4/80 (123115, Biolegend), respectively, on ice for 15 min (3 × 10⁶ cells/sample). The sample were washed three times and resuspended in 200 uL of cold PBS containing 2% FBS and 1 mM EDTA for analysis using flow cytometry (FC; CytoFLEX, Beckman Coulter, USA).

For in vitro electroporation detection, we utilized PI (Invitrogen) staining. PI (640905, BioLegend) was added to the cell suspension simultaneously or after electroporation at 10 μg/mL. After incubation for 15 minutes, the transfection efficiencies of the samples were measured. To determine apoptosis, after electroporation, the resuspended tumor cells were stained with Annexin V-FITC/PI (640905, BioLegend), and analyzed by FC. To analyze the polarization of macrophages, the macrophages were stained with FITC-conjugated anti-human HLA-DR (11–9956-42, eBioscience, San Diego, USA), PE-conjugated anti-human CD206 (12–2069-42, eBioscience), PE-conjugated anti-human CD163 (333606, Biolegend), PE-conjugated anti-mouse CD16/32 (101307, Biolegend), and APC-conjugated anti-mouse F4/80 (123115, Biolegend) .