Fitc conjugated cd25

Leukocytes were isolated from serially collected peripheral blood samples, spleens or liver grafts. Splenocytes were isolated using standard protocol. To obtain single cell-suspensions from cardiac or liver grafts, tissue was cut into small pieces (2 mm) and digested with collagenase IV (Worthington Biochemical Corporation, Lakewood, NJ). The resulting suspension was run through a 70 μm filter and washed with PBS. After centrifugation, the leukocytes were purified using lymphocyte separation medium (Fisher Scientific, Pittsburgh, PA). Phenotypical analysis was performed by using several panels of fluorescein-labeled anti-mouse mABs (against CD3, CD4, CD8, Ly6G, Ly6C, MHCII, CCR2, CXCR1, CD80, CD86) or rat monoclonal antibodies (CD3, CD4, or CD8). All antibodies were purchased from BD Biosciences. Frequencies of T-regulatory cells were identified using APC conjugated CD4, FITC conjugated-CD25, and PE conjugated-Foxp3 (Biolegend) monoclonal antibodies according to the manufacture instructions. Staining was performed with antibodies above (1 μg/10⁶ cells) at 4 °C for 30 min, washed, and analyzed by FACS (BD Biosciences).

HIV-1 BaL was obtained from the NIH AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, NIH. The HIV-1 BaL strain has been shown to efficiently infect, via the CCR5 co-receptor, and replicate within CD4⁺ T-cells^{49 (link)}. Argonaute-1, p-NFκB, NFκB, p-ERK, p-Src, p-Akt, NFAT1, p-CREB, CREB, Oct-1, Ubiquitin, and β-Actin antibodies were obtained from Cell Signaling Technology (Danvers, MA). GW182, D1DR, D2DR, D3DR, D4DR, Sigma-1 Receptor, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). FITC-conjugated p24 GAG (6604665) antibody was obtained from Beckman Coulter, Inc. (Brea, CA). PE-conjugated CD45RO, PE-conjugated HLA-DR, FITC-conjugated CD45RA, FITC-conjugated CD25, FITC-conjugated CD69, PE isotype control, and FITC isotype control antibodies were purchased from Biolegend (San Diego, CA). p-NFAT1 antibody was obtained from Thermo Fisher Scientific (Waltham, MA). Fluo-4, AM was purchased from Invitrogen (Carlsbad, CA). Methamphetamine hydrochloride and sigma-1 receptor inhibitor (BD1047) were purchased from Sigma Aldrich (St. Louis, MO).

The frequency of Th17 and Tregs in CD4+ T cells in peripheral blood were determined with the use of flow cytometry as described previously with some modifications (Chi et al., 2007 (link), 2010 (link)). Briefly, 200 μl of fresh peripheral blood obtained from caudal vein of each rat. The red blood cells were lysed and washed with PBS. Cells were initially stained extracellularly with use of phycoerythrin (PE) anti-human CD4 (eBioscience, United States) at 4°C for 20 min. Subsequently, cells were fixed and permeabilized, and stained with fluorescein isothiocyanate (FITC)-conjugated IL-17A (BioLegend, United States) for Th17 detection and PerCPCy5.5-conjugated IFN-γ (BioLegend, United States) for Th1 detection. For Tregs, the CD4-PE cells were stained with FITC-conjugated CD25 (BioLegend, United States). Flow cytometric analysis was performed with use of a fluorescence-activated cell sorter Calibur cytometer. Data were analyzed with CellQuest software (Becton Dickinson, United States).