Tag it violet proliferation and cell tracking dye

The cells from naïve C57BL/6J mice were first labeled with Tag-it Violet™ Proliferation and Cell Tracking Dye (Biolegend, San Diego, CA, USA), and sorted using Dynabeads™ Untouched™ Mouse T Cells Kit (Invitrogen, California, USA) for getting CD3⁺ T cells. The labeled CD3⁺ T cells were activated with anti-CD3/CD28 beads (ratio 2:1) in 96-well plates at 2×10⁵ cells/well for 4 days with/without HCQ (20, 40, or 60 µM).

“Minipool” cultures of 10–50 transduced B cells were established in 96 well flat-bottom tissue culture plates supplemented by addition of 4 × 10³ irradiated CD40L-L cells and 50 ng/ml IL-21 twice weekly. Supernatants were harvested after 14 days culture and the antibody concentrations were assessed by IgG and IgA ELISA (Cambridge Biosciences). Supernatants were screened by intracytoplasmic staining of PRRSV-1 Olot/91-infected MARC-145 cells (20 (link)). Following secondary labeling with biotinylated IgG (H&L) pAb (Cambridge Biosciences)/streptavidin-Brilliant Violet 421 or Alexa Fluor 647-conjugated IgG mAb (Cohesion Biosciences, London, UK), cells were analyzed by flow cytometry. Supernatants which stained infected cells were subsequently retested by staining both uninfected and infected cells. Selected supernatants were also simultaneously screened for PRRSV-specific reactivity by staining a mixture of infected and uninfected cells, the latter of which had previously been labeled with 10 μM Tag-it Violet Proliferation and Cell Tracking Dye (BioLegend) (21 (link)). Supernatants were additionally screened for neutralizing activity against PRRSV-1 Olot/91 as described above.

PBMCs were thawed at 37°C for 5 minutes, washed and resuspended in complete RPMI. Cells were counted on a hemocytometer using trypan blue to exclude dead cells, then plated at a density of 200,000 cells/well in a 96-well round bottom plate. For intracellular cytokine analysis, cells were stimulated with 1X of BioLegend Cell Activation Cocktail (81 nM Phorbol-12-yristate 13-acetate (PMA); 1.34 μM Ionomycin). After 1 hour, brefeldin A (BD GolgiPlug Protein Transport Inhibitor; 555029) was added and cells were analyzed 18 hours later by flow cytometry using methods described above. For proliferation analysis, PBMCs were stained with Tag-it Violet™ Proliferation and Cell Tracking Dye (625 nM, BioLegend 425101) for five minutes at 37°C and washed with cold media. T cells were then negatively enriched using MojoSort™ Human CD3 T Cell Isolation Kit (BioLegend 480131). Cells were stimulated with DynaBeads (ThermoFisher 1161D) at 37°C for 72 hours in the presence or absence of inhibitors as indicated.

Freshly isolated PBMC were stained using Tag-it Violet Proliferation and Cell Tracking Dye (Biolegend, USA) according to the manufacturer's instructions. Briefly, total PBMC were adjusted to 1 × 10⁷ cells/ml in PBS and incubated with the Tag-it Violet Proliferation and Cell Tracking Dye at a concentration of 5 μM. Cells were incubated for 20 min at 37°C in the dark. Quenching was done by addition of cell culture media supplemented with 10% FCS. After washing, stained cells were used for visualization of iNKT cell proliferation after stimulation with αGC.