Bv421 anti flag

WT or ligand-binding defective (ΔLB, G303E) forms of human RARα ^{45 (link)} with a N-terminal FLAG-tag were cloned into pMSCV-Thy1.1 retroviral vectors. Retroviral supernatants containing viral particles were produced from plasmid-transfected Platinum E cells. Naïve T helper cells were cultured for 16 h in cRPMI with plate-bound anti-CD3 (5 μg/mL) and soluble anti-CD28 (2 μg/mL), then transduced with retroviral supernatants in the presence of polybrene (8 μg/mL) by spin transduction (90 m, 2300 rpm, 32^oC). Cells were rested for 1–2 h after transduction, then reactivated in a Th17-polarizing condition with or without 10 nM RA for 20 h. Cells were then harvested and stained for surface Thy1.1 (clone OX-7), fixed with 1% paraformaldehyde for 1 hour, permeabilized with flow cytometry perm buffer (Tonbo Biosciences; San Diego, CA) and stained with BV421 anti-FLAG (BioLegend, clone L5) and DRAQ5. Cells were spun onto Cell-Tak coated glass slides and imaged with a Nikon A1 laser scanning confocal microscope with 60× objective. Cellular localization of FLAG-RARα was analyzed using ImageJ.