Olytetrafluoroethylene We utilized Transwell -COL collagen-coated pore polytetrafluoroethylene memmembrane insert (Sigma-Aldrich) to prepare an in vitro BBBas described previously [27]. brane insert (Sigma-Aldrich) to prepare an in vitro BBB model model as described previouslyMouse model: Initial, we made use of mouse endothelial and astrocytic-cells to represent our [27]. Mouse model: First, we utilized mouse endothelial and astrocytic-cells to represent our future Farnesyl Transferase site proposed perform with the HIV mice model to study the pharmacokinetics, tissue future proposed work using the HIVon viral suppression. Briefly, the mouse astrocytes distribution, and efficacy of Cur-D mice model to study the pharmacokinetics, tissue distribution, and efficacy of Cur-D onthe bottom of 12-well plates. mouse24 h of adhe(2 105 cells/well) were NOD2 drug seeded on viral suppression. Briefly, the After astrocytes (two 105 cells/well) were seeded around the 105 cells/well) have been seeded onto the upper sidemouse sion, mouse endothelial cells (2 bottom of 12-well plates. After 24h of adhesion, on the endothelial -cells (2 105 cells/well) were had been placed inside a 12-wellside ofcontaining astroTranswellCOL inserts, and also the inserts seeded onto the upper plate the Transwell OL inserts, cells the inserts the BBB model and were grown for 5 daysastrocytes. These cytes. These and constitute were placed inside a 12-well plate containing to attain 90 cells constitute the BBB model and have been grownupper inserts containing endothelial cells confluency. Right after attaining 90 confluency, the for 5 days to attain 90 confluency. Following reaching 90 the wells containing U1-differentiated macrophages. Transendothewere transferred to confluency, the upper inserts containing endothelial cells were transferred to the wells containing U1-differentiated macrophages. Transendothelial Precision lial electrical resistance (TEER) using EVOM2 Epithelial Voltohmmeter (Globe electrical resistance (TEER) usingFL) was measured as described [27]. A mean TEER Instruments, Instruments, Sarasota, EVOM2 Epithelial Voltohmmeter (Planet Precision worth of one hundred to 120 Ohms was measured as described [27]. A imply model and of one hundred to 120 our preSarasota, FL) cm2 was observed in the confluent BBBTEER value published in Ohms vious reports [27]). the confluent BBB model of Cur-D on CSC-induced viral replication, cm2 was observed inTo decide the efficacy and published in our preceding reports [27]). endothelial cells efficacy of Cur-D on CSC-induced to a replication, endothelial cells in To ascertain the inside the upper inserts had been exposed viralsingle dose of control (DMSO), CSC (40 /mL), Cur-D (0.four ), single dose /mL) (DMSO), CSC (40 /mL), Curthe upper inserts have been exposed to aand CSC (40of control+ Cur-D (0.four ) and observed for three days. and CSC (40 /mL) + Cur-D (0.4 of CSC, observed for 3 CSC dose shows D (0.4 ), Within this case, we utilised a greater dose ) andbecause a lowerdays. In this case, inability to cross dose of and due to the fact a suppress HIV across the BBB. HIV-1 viral loads we utilized a higher the BBBCSC, effectivelylower CSC dose shows inability to cross the BBB were measuredsuppress HIVthe cell culture supernatant in the bottom chamber making use of a and effectively daily in across the BBB. HIV-1 viral loads had been measured every single day p24 ELISA kit. within the cell culture supernatant in the bottom chamber making use of a p24 ELISA kit. Huma model: After establishing the impact of Cur-D against CSC-induced HIV repliHuma model: Soon after establishing the effect of Cur.
