Anti cd4 pe clone rm4 5

BALB/c mice (n = 8–10) immunized with pE1D2 or pcTPANS1 were depleted from CD4⁺ or CD8⁺ cells upon inoculation of in-house produced ascitic fluids containing anti-CD4 (clone GK 1.5) or anti-CD8 antibodies (clone 56-3.7) (39 (link)). Animals were inoculated by the intraperitoneal route (i.p.) with 20 μL of ascitic fluids on days 4 and 2 before DENV2 challenge. T cell depletion was monitored by flow cytometry in blood cells stained with anti-CD3 FITC (clone 145-2C11), anti-CD8 PerCP (clone 33-6.7) and anti-CD4 PE (clone RM4-5) antibodies (BD Biosciences), and evaluated on FlowJo software.

Cell suspensions from vaginal tissues were prepared as follows: tissues were cut into small pieces and treated with Liberase TL (Roche, Indianapolis, IN, USA) in MEM medium at 37 °C for 40 min. Tissues were next pressed through a 70 µm cell strainer and washed in PBS/1%FBS. Cell suspensions were pretreated with the Fc receptors block-rat anti-CD16/32 antibody (2.4G2) (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. The following antibodies were used: anti-CD3-FITC (145-2C11, ThermoFisher Scientific), anti-CD4-PE (clone RM4-5, BD Biosciences), anti-CD8-PerCP (clone 53-6.7., BD Biosciences), anti-NK1.1-APC (clone PK136, BD Biosciences), anti-CD11b-FITC or PE (clone M1/70) (BD Biosciences), anti-CD69-APC antibody (H1.2F3, eBioscience), anti-CD11c-APC (HL3, eBioscience), rat anti-F4/80-FITC (BM8, eBioscience) and rat anti-I-A-PE (M5/114.15.2, eBioscience) antibodies, and rat anti-Gr-1-PE (RB6-8C5, BD Biosciences). Following the immunolabeling for the extracellular markers, cells were fixed with Perm/Wash buffer (BD Bioscience) and were incubated with anti-IFN-γ APC (eBioscience, clone-XMG1.2). The stained cell suspensions were analyzed in FACS Calibur for the percentage of positively stained cells and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).