Zombie amine dye

The 3 × 10⁶ PBMCs from healthy donors or individuals carrying heterozygous IKZF1 mutation were cultured in the presence poly(I:C) (10 μg/ml, Invivogen), LPS (5ng/ml, Sigma), CL075 (1 μg/ml, Invivogen) and CpG (ODN 2216, 7.5 μΜ, Invivogen) with or without IFN-α (3000 IU/ml, R&D), with or without anti-CD303 and anti-CD304 (Biolegend), with or without 0.1, 1 or 10 μM lenalidomide (Sigma). Cells were cultured for 14 h at 37 ^oC, 5% CO₂, with addition of Brefaldin A (10 μg/ml, eBioscience) after 3 h. For dead-cell exclusion (usually <30%) cells were stained with Zombie amine dye (Biolegend), surface markers and then intracellular cytokines antibodies after fixation and permeabilization (eBioscience), as above.

For cytokine production, PBMC from healthy controls or in vitro-generated cells were cultured in the presence of polyinosinic:polycytidylic acid (poly(I:C), 10 μg/ml, Invivogen), Lipopolysaccharide (LPS, 5ng/ml, Sigma), CL075 (1 μg/ml, Invivogen) and CpG (ODN 2216, 7.5 μΜ, Invivogen) for 14h at 37°C, 5% CO₂ with addition of Brefeldin A (10 μg/ml, eBioscience) after 3 hr. Dead cells (usually < 30%) were excluded with Zombie amine dye (Biolegend). Intracellular cytokine staining was performed after surface staining, fixation, and permeabilization (eBIoscience) according to manufacturer’s instructions.
For T cell proliferation, FACS-purified ex vivo or in vitro generated DCs (2,500-8,000 DC/well) were cultured with FACS purified allogeneic CD3⁺ T cells at a ratio of 1:10 DC:T cell (n = 2-9 DC/T cell pairs). Positive controls were generated by T cell co-culture with CD3+CD28+ beads (Dynabeads®, Thermo Fisher Scientific) at T cell:bead ratio 1:1. T cell proliferation was assessed by CFSE dilution on day 5 of culture.