Zombie redtm fixable viability kit

The pcDNAswCD40 and pcDNAboCD40 constructs were used to transfect HEK-293A cell monolayers using Polyethylenimine (Polyscience) as previously described [33 ]. Following 48 hr. incubation, one million transfected cells were added to each well of a 96 well V-bottom plate and stained with Zombie Red^TM Fixable Viability Kit (Biolegend) following the manufacture's protocol. The pcDNAswCD40 and pcDNAboCD40 transfected HEK-293A cells were incubated with 5μg/mL of the mAb 2E4E4 or 5μg/mL of IgG1 isotype control for 30 min. and washed 3X with blocking buffer (cDMEM with 0.05% sodium azide/20% bovine Serum). The cells were incubated for 30 min. with AffiniPure F(ab’)₂ Fragment Donkey Anti-Mouse IgG (H+L) conjugated with FITC (Jackson ImmunoResearch Laboratories, INC.), washed 3X with block buffer, and stored in FACS fixer (12.5% formaldehyde/PBS). Data was collected using BD FACScalibur™ (Becton Dickinson) and data analysis was done using FlowJo 10 software (FlowJo).

Splenocytes were isolated from transduced recipients and labeled with CellTrace Violet using the CellTrace™ Violet Cell Proliferation Kit (Life Technologies, Carlsbad, CA). Four and a half million cells/well were cultured in flat bottom 96-well plates (BD Falcon, Franklin Lakes, NJ) with 300 μl of completed RPMI-1640 media containing 50 ng/ml OVA (Sigma-Aldrich, St. Louis, MO) for 96 h. Recombinant human factor IX (rhF9) was used as an unrelated antigen control. Concanavalin A [ConA, a mitogen to stimulate non-specific T cell proliferation 58 (link)) (Sigma) was used as a positive control for CD4 T cell proliferation. After culture, cells were collected and stained with anti-CD4, anti-CD45.2, and anti-Foxp3 antibodies. Zombie Red^TM Fixable Viability Kit (BioLegend) staining was used to exclude dead cells. Cells were analyzed by LSRII flow cytometry (BD Biosciences), and data were analyzed using FlowJo software.