HIV seroconversion final results in preexposure prophylaxis (PrEP) trials of oral tenofovir (TFV)-containing regimens are highly sensitive to drug concentration, yet less-than-daily dosing regimens are under study. followed by a terminal 48?h (38C76) half-life; for 25?min at 28C. Plasma was isolated (1?ml) from the CPT tube, and then the PBMCs were collected from the buffy coat and washed twice with phosphate-buffered saline (PBS). Cells were resuspended in 1?ml PBS for cell counting. Cell pellets were lysed with 70% methanol and stored at ?80C until analysis. Colon samples Colon samples were collected through a flexible sigmoidoscope (Evis Exera, Olympus America Corp., Center Valley, PA). Endoscopic brushes (Kimberly Clark, Roswell, GA) were used to collect superficial colonic fluid and cells.9 Brushes were luminal and weighed fluid was eluted using 1? ml centrifugation and PBS at 800g, 10?min, 4C. Examples had been kept at instantly ?80C. Brushes had been later on dried and weighed to determine the original brush, and hence sample, weight. Thirty biopsies were collected using 3.7-mm pinch biopsy forceps (Microvasive no. 1599; Boston Scientific Corp., Natick, MA) 10C20?cm from the anus, and placed in RPMI medium with 10% fetal bovine serum (R10 media) until processing.10 Four colonic biopsies were weighed and homogenized in 500?l ice-cold 70% methanol; samples were immediately frozen at ?80C. The remaining biopsies were used for enzymatic extraction of mucosal cells. Vaginal sampling Samples were scheduled not to coincide with menses. CVL was performed with 10?ml of Normosol-R (Hospira, Lake Forest, IL) repeatedly applied against the cervix and vaginal walls for 30?s and collected into a 10-ml syringe with Luer-Lok tip. CVL was transferred into a 15-ml conical tube and centrifuged at 450g, 10?min, 4C to pellet CVL cells.11 Supernatant was aliquoted into 1-ml fractions for analysis. The CVL pellet was washed twice and lysed in 70% methanol. For vaginal biopsies, a speculum was inserted into the vagina and five vaginal biopsies were taken with 2.34.2-mm Tischler gold-plated gynecological forceps. One biopsy was homogenized and flash frozen similar to the colon biopsies. The remaining biopsies were used for cell extraction. Tissue cell extraction To release cells for intracellular analysis from colonic and vaginal tissue, biopsies were incubated with a dissociative enzyme cocktail consisting of Sema3d collagenase (0.5?mg/ml, Sigma-Aldrich, St. Louis, MO), DNase I (0.083?U/ml, Roche, Indianapolis, IN), elastase (0.07?U/ml, Worthington Biochemicals, Lakewood, NJ), and hyaluronidase (0.4?U/ml, Worthington Biochemicals, Lakewood, NJ). The digestions were carried out in RPMI with 7.5% FBS in 50?ml-conical tubes at 37C with agitation (Invitrogen, Carlsbad, CA) essentially as described.11 Cells were counted using Guava/Millipore EasyCyte Plus (Millipore, Billerica, MA). Cell separation with MACS CD4 cells were isolated via positive selection with CD4 microbeads using magnetic affinity column separation (MACS) according to the manufacturer’s recommended protocol (Miltenyi Biotec, Auburn, CA). CD4-positive and CD4-negative fractions were collected for cell counting and intracellular drug analysis. To maximize cell 1029877-94-8 yield, we did not take aliquots to test CD4 cell purity. In our prior studies, PBMC CD4 purity is 95% and tissue cell extraction CD4 purity is 75C85%. Drug concentration analysis Accelerator mass spectrometry (AMS): Sample preparation: 14C analysis of neat plasma, tissue homogenate, colonic fluid, and CVL samples for TFV concentration was performed at the Massachusetts Institute of Technology (MIT) Biological Engineering Accelerator Mass Spectrometry (BEAMS) lab.12,13 For TFV-DP evaluation, the isolation treatment useful for LC-MS/MS recognition of TFV-DP (described below) was used ahead of AMS recognition. Samples had been reconstituted in 50?l 0.5% acetic acid in water and analyzed using AMS. No 13C-TFV inner standard was put 1029877-94-8 into these examples, as there is concern that 13C in examples would raise the history in each test, decreasing the sensitivity of detection of 14C thereby. Accelerator mass spectrometry (AMS): Test evaluation Aliquots of cell lysates or luminal liquid examples (1C2.75?l) were useful for AMS evaluation. Samples had been adsorbed onto CuO natural powder pellets made by contact with an O2 atmosphere in vacuum pressure oven. Pellets had been used in a laser-induced combustion user interface for AMS evaluation. Five samples had been follow a quantitation regular of 0.0030 dpm/l. The combustion user interface generates CO2, which can be then sent to the ion resource and used to look for the total 14C in the sample. The TFV-DP lower limit of quantitation was 1.32?fmol/million cells based on a 1.5-l sample. The TFV lower limit of quantitation was 0.09?ng/ml based on a 1.5-l plasma sample. The TFV data showed a subject-dependent (analytical run) difference between the AMS and LC-MS/MS concentrations, ranging from 1- to 1029877-94-8 6-fold. To adjust for this, we scaled the AMS TFV results using individual LC-MS/MS results on the same sample as a reference. LC-MS/MS Plasma samples from several subjects were also assayed for TFV and TFV-DP for comparison.