Anti cd4 pe rpat4

The CFSE Cell Division Tracker Kit (Biolegend) was used for flow cytometry analysis of in vitro CD4⁺ T cells proliferation assay according to manufacturers' protocol. Briefly, autologous lymphocytes were pre-treated with 0.1 μM CFSE before being added to MDDC cultures in the presence of the lymphocyte mitogen Concanavalin A (5 μg/mL; Sigma Aldrich, Merck) for 120 h (32 (link)). At the end of assay, cells were then labeled for anti-CD3 APC (MEM-57) and anti-CD4 PE (RPAT4) (BD Biosciences). The Live/Dead Fixable Cell Stain Kit was added to the assay according to the manufacturer's instructions. Cells were then washed twice with PBS and resuspended in 200 μL of 4% Formaldehyde-PBS to proceed to flow cytometry analysis as above-mentioned.

iCD4 cells were prepared as previously described and NK cells were isolated and stimulated with iCD4 according to a previously established protocol [40 (link)]. Briefly, iCD4s were plated at NK:iCD4 ratios of 10:1 or alone for 10 days in R10-IL-2. Cells were collected and stained with Aqua amine reactive fluorescent dye (Invitrogen, Burlington, ON, Canada) to determine viability and cell-surfaced stained with anti-CD3-APC-eFluor 780 (UCHT1; eBioscience) and anti-CD4-PE (RPA-T4; BD Biosciences, Mississauga, ON, Canada). p24 staining, acquisition, and analysis were performed as described above.

Where indicated, cells were first stained with Live/Dead Fixable near-IR (Invitrogen, Eugene, OR, USA). Human cells were stained with anti-HLA-DR FITC (G46-6), anti-CD11c PE (B-ly6), anti-CD80 PE-Cy7 (L307.4), anti-CD40 APC (5C3), anti-CD86 BV510 (FUN-1), anti-CD123 BV421 (9F5), CD3 FITC (UCHT1), anti-CD4 PE (RPA-T4), or CD8 PE-Cy7 (RPA-T8) (all BD Biosciences). Mouse cells were stained with anti-IL-17 A PE (TC11-18H10), anti-CD8a V450 (53-6.7) anti-CD40 FITC (3/23) anti-CD80 APC (16–10A1), anti-CD11b BV510 (M1/70) (all BD Biosciences); anti- IFN-γ APC (XM61.2), anti-CD11b PE-Cy7 (M1/70), anti-CD4 PE-Cy7 (RM4–5), anti-CD4 PE (RM4–4) was used when anti-CD4⁺ mAb was used to deplete CD4⁺ cell, anti-CD45 PerCP (30-F11), anti-CD90.2 PerCP (30-H12) (all BioLegend San Diego, CA); anti-TNF FITC (MP6-XT22), anti-CD90.2 FITC (54-2.1) anti-Granzyme B PE (NGZB), anti-B220 APC (RA3-6BC), anti-CD11c PE-Cy7 (N418), anti-CD90.2 PerCP-eF710 (30-H12), anti-CD11c PE (N418), or anti-MHC-II PE-Cy7 (M5/114.15.2) (all eBioscience, San Diego, CA). Samples were run on a FACSCanto II (BD Biosciences, San Jose, CA, USA), and analyzed using FACS Diva (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland OR). Fluorescence minus one controls were used to define costimulatory molecule expression by DCs stimulated with poly I:C or chlamydial antigen.