P24 elisa kit

Chimeric HIV virus EcoHIV/NDK was employed for mouse infection. EcoHIV/NDK was generated by replacing the viral gp120 protein with gp80 for the ecotropic moloney murine leukemia virus^{82 (link)}. The viral plasmid was a kind gift from Dr. David Volsky (Icahn School of Medicine at Mt. Sinai, New York, NY). Viral stocks were prepared by transfecting HEK 293 T/17 cells with the EcoHIV/NDK plasmid using lipofectamine 2000 (Invitrogen). Viral concentration in the filtered supernatant was quantified by p24 ELISA kit (Zeptometrix).

Modified TZM-bl cells were infected with HIV-1_NL4-3 at an MOI of 0.5 or 1 for 6 h, then washed three times with PBS and cultured in complete DMEM medium for 3 days. Cells were then washed once with PBS and lysed in 100 µl of lysis buffer (Promega). Luciferase activity in 30 µl of cell suspensions was measured by a BrightGlo Luciferase assay according to the manufacturer’s instruction (Promega). The cultured supernatants were collected at indicated days post-infection, HIV-1 p24 was determined using a p24 ELISA kit (ZeptoMetrix).

Lentivirus containing a construct consisting of the human arrestin promotor (HXARR3), enhanced Halorhodopsin (eNpHR) from Natronomonas and Enhanced Yellow Fluorescent Protein (EYFP), LV-HXARR3-eNpHR-EYFP, was injected (2.5 µL; titer: 5730 ng/µL; p24 Elisa kit, ZeptoMetrix Corp., New York, NY, USA) between the two plexiform layers of cultured human retinal explants at four different locations, then the retinas were cultured for at least an additional 48 h before experimentation.

5x10⁵ chronically HIV-infected H9 T-cells were exposed once or daily to 5 or 50 μM curcumin or serum-free RPMI as control At several time points post-treatment, supernatants were collected and HIV-1 p24-antigen was measured using a commercial p24 ELISA kit (Zeptometrix Corp., Buffalo, NY, USA), as per the manufacturer’s instructions. Alternatively, 1x10⁵ chronically infected H9 T-cells were treated with 5 or 50 μM curcumin for 1 hour and subsequently exposed to TLR ligands for 24 hours. TLR ligands included the TLR3 ligand poly I:C (25 μg/mL; Sigma-Aldrich), the TLR4 ligand LPS from Escherichia coli (100 μg/mL Sigma-Aldrich) and the TLR5 ligand flagellin from Salmonella typhimurium (10 μg/mL; Alpha Diagnostic, San Antonio, TX, USA). TLR ligand concentrations were selected according to previous studies, described elsewhere [5 (link), 19 (link), 33 (link)].