Penicillin streptomycin

Human umbilical cord matrix-derived stem cells (UCM-MSCs) were provided by the Asan Stem Cell Center (Asan Institute for Life Sciences, Seoul, Korea). The cells were obtained from the previously described protocols [23 (link)]. Both stem cell types (UCM-MSCs and AM-MSCs) were cultured on 0.1% gelatin-coated culture dishes with a culture medium. The culture medium was based on DMEM/F12 supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA), 10 ng/ml fibroblast growth factor 2 (FGF2; Peprotech, Rocky Hill, NJ, USA), 1% NEAA (Gibco), 1% Penicillin/Streptomycin (GeneDirex). Cells were passaged 1 to 3–5 every 3 to 4 days using trypsin/EDTA (Gibco, NY, USA).

A phagocytosis assay was conducted as previously described (47 (link)). J774A.1 murine macrophages (ATCC CCL-240) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GeneDireX) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 100 U/mL penicillin-streptomycin (GeneDireX), and cells were incubated at 37°C with 5% CO₂. J774A.1 cells were stimulated with 100 nM phorbol myristate acetate (PMA; Sigma-Aldrich) for 3 days. The bacterial pellet was washed twice with PBS, resuspended in DMEM, and used in the assays. Reactions were initiated by incubating 2 × 10⁵ CFU (multiplicity of infection [MOI] = 2) of E. anophelis C08 with heat-inactivated (56°C for 30 min) test serum and nOMVs/iOMVs in the wells. After a 2-h incubation with gentle shaking (80 rpm), the samples were serially diluted and plated. Bacterial killing rates were determined by comparing the number of reduced CFU with those observed using PBS-immunized antiserum. Hyperimmune serum from OMV-immunized mice was used as a positive control (data not shown, animal studies were approved by the National Defense Medical Center Institutional Animal Care and Use Committee, NDMC IACUC-19-250).

The opsonophagocytosis assay was conducted as previously described (18 (link)). Briefly, J774A.1 murine macrophages (ATCC CCL-240) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), GeneDireX supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), and 100 U/mL penicillin-streptomycin (GeneDireX). Cells were incubated at 37°C with 5% CO₂. The bacterial pellet was washed twice with PBS and resuspended in DMEM before the assays. Reactions were initiated by incubating 1 × 10⁵ or 2 × 10⁵ CFU (multiplicity of infection = 1:1 or 1:20) of E. anophelis C08 with heat-inactivated test serum and iOMVs in the wells. After a 2-h incubation with gentle shaking (80 rpm), the samples were serially diluted and plated. Bacterial killing rates were determined by comparing the difference in CFU between the iOMV-immunized serum and PBS-immunized antiserum treatments. Hyperimmune serum from OMV-immunized mice was used as the positive control.