Fitc conjugated cd45ra

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood by density gradient centrifugation on Ficoll-Paque PLUS (Cytiva). The isolated PBMCs were then analyzed with an 8-color flow cytometer (FACSCanto™II, BD Biosciences, CA, USA) after labeling with the following anti-human fluorochrome-conjugated antibodies: FITC-conjugated CD38 (clone HIT2); APC or FITC-conjugated PD-1 (clone EH12.2H7); FITC-conjugated CD45RA (clone HI100); PE-conjugated CD24 (clone ML5); PE-conjugated ICOS (clone C398.4A); PE-conjugated CCR6 (clone G034E3); PerCP-conjugated CD3 (clone UCHT1); PE/cyanine7-conjugated CD27 (clone O323); PE/cyanine7-conjugated CD4 (clone OKT4); APC-conjugated CD19 (clone HIB19); APC-conjugated CD45RO (clone UCHL1); APC-conjugated CXCR3 (clone G025H7); and Brilliant Violet 421-conjugated CXCR5 (clone J252D4) (all antibodies were from BioLegend, CA, USA). Cell viability was assessed using a live/dead assay (Fixable Viability Dye eFluor 780; eBioscience). All data were collected and analyzed using FlowJo software. The gating strategies of the isotype-matched IgG control for specific cell populations are shown in the supplementary figure (Figure S1).

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).