Fluorescence conjugated antibodies

Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Murine breast cancer cells (E0771) were obtained from CH3 BioSystems (Buffalo, NY). All cell lines were routinely grown in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD), and an antibiotic/antimycotic solution at final concentrations of 100 U/ml penicillin, 100 μg/ml streptomycin and 0.250 μg/ml amphotericin B (Sigma-Aldrich, St. Louis, MO). Cells were maintained in T-25 tissue culture flasks (Techno Plastic Products AG, Trasadingen, Switzerland) in a standard humidified incubator with 5% CO₂ balanced-air at 37°C. Prior to experimentation, RAW264.7 cells were serum deprived. Fluorescence-conjugated antibodies purchased from BioLegend (San Diego, CA) were employed to identify macrophages (CD45-FITC, clone30-F11) and to determine their phenotype (CD86-PerCP/Cy5.5 -clone GL-1- for M1 phenotype and CD206-APC -clone C08C02- for M2 phenotype) by fluorescence-activated cell sorting (FACS).

CpG 1018 was custom-synthesized by Trilink Biotechnologies (San Diego, CA, USA). AddaVax was obtained from InvivoGen (vac–adx–10, San Diego, CA, USA). Reagents used in molecular cloning, such as Phusion DNA polymerase and restriction enzymes, were purchased from New England Biolabs (NEB, Ipswich, MA, USA). Fluorescence-conjugated antibodies used in immunostaining and flow cytometry were obtained from BioLegend (San Diego, CA, USA). TMB substrate was purchased from Thermo Fisher Scientific (34028, Waltham, MA, USA). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibody was purchased from Cytiva (NA931, Marlborough, MA, USA). HRP-conjugated anti-mouse IgG1 was purchased from Invitrogen (046120, Waltham, MA, USA). HRP-conjugated anti-mouse IgG2c was purchased from Bethyl Laboratories (A90–136P, Montgomery, TX, USA).

Cells were stained with Fixable Viability Dye eFluor 506 (1:1000) in 1x PBS for 30 min at 4°C (Thermo Fisher Scientific, USA) before incubated with Fc block (BioLegend, USA) for 15 min at 4°C. Cells were stained with fluorescence-conjugated antibodies (BioLegend, USA) diluted in FACS buffer and incubated on ice for 30 min in the dark. Macrophages were identified as CD45+ Ly6G- Gr1- F4/80+ CD11b+ cells. Cells were analyzed on a BD LSRFortessa (BD Biosciences, USA) or Attune NxT Flow Cytometer (Thermo Fisher Scientific, USA). Data analysis was performed using FlowJo software (FlowJo, USA).