Apc conjugated anti mouse f4 80

We resuspended the macrophages in auto MACS Rinsing Solution. Nonspecific Ab binding was blocked using an Fc blocking Ab, and the purity of isolated macrophages was determined by staining with APC-conjugated anti-mouse F4/80 (BioLegend). The surface markers of macrophages were evaluated using PE/CY7-conjugated anti-mouse CD86 (Thermo). To stain CD206 intracellularly, macrophages were fixed and incubated with PE-conjugated anti-mouse CD206. Flow cytometry was performed using a FACSCalibur cytometer (BD Biosciences) and analyzed with FlowJo software.

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) .

Spleen and colonic tumor were isolated into single-cell suspensions. After
washing, the single cells were pre-incubated with the fluorescence-conjugated
primary antibody for 30 minutes at 4°C in the dark. Then, we performed flow
cytometry on a FACS Calibur flow cytometer (BD Biosciences, USA). The results
were analyzed by FlowJo software. The following antibodies were used:
FITC-conjugated anti-mouse/human CD11b (BioLegend, #101206, USA), APC-conjugated
anti-mouse Ly-6G/Ly-6C (Gr-1) (BioLegend, #108412, USA), APC-conjugated
anti-mouse CD3 (BioLegend, #100235, USA), FITC-conjugated anti-mouse CD4
(BioLegend, #100405, USA), APC-conjugated anti-mouse F4/80 (BioLegend, #157306,
USA), FITC-conjugated anti-mouse CD11c (BioLegend, #117305, USA), APC-conjugated
anti-mouse I-A/I-E (BioLegend, #107614, USA).

Single cell suspensions from the peritoneal lavages and visceral adipose tissues were plated in RF10c media with the addition of GolgiPlug (containing Brefeldin A) and GolgiStop (containing Monensin) Protein Transport Inhibitors (BD) according to manufacturer’s protocols. Plates were incubated for 9 h at 37°C and 5% CO₂.
After incubation, cells were washed with PBS and stained for flow analysis. Single cell suspensions were labeled with Fc block (purified anti–mouse CD16/32; BD) for 10 min, followed by labeling with antibodies for 20 min at 4C. Cells were washed twice with a staining buffer consisting of 5% FBS (Gemini Bio-products) in DPBS (Corning). For intracellular staining, the Cytofix/Cytoperm kit (BD) was used per manufacturer’s protocols. After staining, cells were analyzed using a custom configured Fortessa cytometer, and by using FACSDiva software (BD). Data were analyzed using FlowJo software vX (Tree Star). Antibodies used to distinguish macrophages were: PB-conjugated anti–mouse CD45, BV711-conjugated anti–mouse CD11b, BV605- and PE-conjugated anti–mouse CD206, BV785-conjugated anti–mouse CD19, APC-conjugated anti–mouse F4/80, and PE-Cy7–conjugated anti–mouse TNF (BioLegend).