Hepes

Female C57BL/6J mice (8–12 weeks old) were purchased from Jackson Laboratory and maintained in specific-pathogen-free conditions. All procedures were approved by the Institutional Animal Care Committee of the University of Pittsburgh (IACUC). To generate bone marrow derived macrophages (BMDMs), mouse bone marrow was cultured for 6 days in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 U/ml), 2 mM l-glutamine, and 20 mM Hepes supplemented with recombinant mouse M-CSF (60 ng/ml; R&D Systems). BMDMs were scraped with cold PBS and plated in either a 48 or 96 well plate for qPCR and IF, respectively, and rested overnight at 37°C. BMDMs were stimulated on day 7 with the appropriate doses of IL-6 (R&D Systems) and IL-10 (R&D Systems) at the noted time points. In some experiments, cells were treated with 1 nM of Fedratinib 20 minutes prior to cytokine stimulation.

Primary human macrophages were cultivated from purified PBMCs from a healthy donor. One point five million PBMCs/well were plated in RPMI-1640 supplemented with 10% FBS and 1% Pen-Strep on 13 mm circular coverslips in a 24-well plate for 2 h. Non-adherent cells were rinsed off with PBS, and RPMI-1640 supplemented as described and with additional 20 mM Hepes and 25 ng/ml M-CSF (R&D Systems). Medium was changed every third day and the cells were allowed to differentiate for 10 days. Normal human serum was collected and pooled from six healthy donors and stored at −80°C immediately after isolation. After a 1h incubation of CFSE-labeled sorted DAF^lo or DAF^hi GC B cells with macrophages in RPMI-1640, supplemented with 10 µM CaCl₂ and 10 µM MgCl₂ and 10% of either thawed or heat-inactivated human serum, samples were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100, followed by staining with AlexaFluor-546 phalloidin (ThermoFisher). Phagocytosis was quantified by microscopy where phalloidin⁺ macrophages were counted, and macrophages containing CFSE signal and phagocytic vesicles were considered as phagocytosing. A total of 200 macrophages per well were counted.

Details of the isolation and characterization of columnar and squamous primary epithelial cells from endocervical and vaginal biopsy tissues were described before (10 (link)). Written informed consent was obtained from all subjects, and the procedure was performed under the guidelines of a protocol approved by the institutional review board of the University of California at Davis. Anonymous leukopaks or Trima filters of peripheral blood samples were provided by the UCLA Center for AIDS Research. PBMCs were purified on Ficoll gradients by standard techniques. We isolated CD4⁺ T cells from PBMCs with immunomagnetic beads (Stem Cell Technologies). The purity of the isolated cells was assessed by flow cytometry after isolation, and the purity of CD4⁺ cells was >90%. Primary T cells were cultured in RPMI (Life Technologies, Inc.) supplemented with 10% FBS (HyClone), 2 mM l-glutamine, 10 mM HEPES (pH 7.2), 100 μg/ml streptomycin, 100 U/ml penicillin, and 20 U/ml interleukin-2 (R&D Systems).