Blood was collected in BD Vacutainer® CPT™ Cell Preparation Tubes and PBMCs were isolated following the manufacturer’s protocol. Plasma was immediately frozen at − 70 °C in 500 μl aliquots for TFV, medroxyprogesterone acetate (MPA), and progesterone analysis. PBMCs were treated with BioLegend red blood cell (RBC) lysis buffer to minimize interference of RBCs in TFV-DP quantification. Cells counts and viability were determined using trypan blue exclusion on the Countess I (Invitrogen).
Rectal and vaginal tissues biopsies were collected using medical forceps with three pinches collected at each site. Extraneous liquid was carefully removed, and tissue was weighed on an analytical balance; with a median of 15.9 mg of rectal tissue and 20.3 mg of vaginal tissue collected per sample. Due to the small amount of tissue collected from four animals in the DMPA− group in Phase 1, tissues were combined into two pools (two animals each) to ensure sufficient yield of TFV-DP and dATP. This resulted in only two data points for analysis (denoted by triangles inFig. 3A and B ). Biopsies were not combined in any of the subsequent experiments from Phase 1 or 2.
Swabs (Fisherbrand™ Synthetic-tipped Applicator) were inserted in the vagina and rectum for 5 min to ensure uptake of mucosal secretions. The volume of fluid for each sample was determined by weighing the swabs pre- and post-collection.
Rectal and vaginal tissues biopsies were collected using medical forceps with three pinches collected at each site. Extraneous liquid was carefully removed, and tissue was weighed on an analytical balance; with a median of 15.9 mg of rectal tissue and 20.3 mg of vaginal tissue collected per sample. Due to the small amount of tissue collected from four animals in the DMPA− group in Phase 1, tissues were combined into two pools (two animals each) to ensure sufficient yield of TFV-DP and dATP. This resulted in only two data points for analysis (denoted by triangles in
Swabs (Fisherbrand™ Synthetic-tipped Applicator) were inserted in the vagina and rectum for 5 min to ensure uptake of mucosal secretions. The volume of fluid for each sample was determined by weighing the swabs pre- and post-collection.