Polycarbonate membrane insert

Migration assays were performed as previously described [37 (link)]. Cells were homogenized in the absence of plasma for 24 h, and then cultured at 1x10⁵ cells/well in 24-well Transwell plates with polycarbonate membrane inserts (Corning Inc., Corning, NY, USA). For the invasion assays, the membrane was coated with Matrigel (Thermo Fisher Scientific), and cells were cultured at 2x10⁵ cells/well. After incubation for 24 hours, the cells were fixed with 100% methanol and stained with 0.05% crystal violet. Five random fields were counted, and three independent experiments were conducted for each assay.

Human epithelial cells VK2/E6E7 (ATCC CRL-2616) donated by Wuxi People’s Hospital were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), and antibiotics (10 U/mL ampicillin, 10 µg/mL streptomycin) at 37 °C in a 5% CO₂ humidified incubator. To establish human vaginal epithelial monolayer barrier model cells, the apical side (AP) of Transwell plates (Corning, polycarbonate membrane inserts, 0.4 mm pore size) was prewetted with chilled PBS solution. A suspension of 5 × 10⁴ VK2/E6E7 cells in 500 µL DMEM was added to each insert, followed by the addition of 1.5 mL medium to the basolateral side (BL). The plate was then incubated at 37 °C for approximately 21 days to allow the formation of a stable transepithelial electrical resistance (TEER)-based monolayer.
The experimental design included a control group, a model group, and treatment groups with L. delbrueckii DM8909 (DM8909), L. crispatus CCFM1339 (CCFM1339) and L. crispatus FHNXY73M2 (FHNXY73M2). Except for the control group, all other groups received DMEM containing 25 μg/mL lipopolysaccharide (LPS, Escherichia coli 055: B5, Sigma) for 24 h of intervention. Subsequently, the cells were washed with PBS and further treated with DMEM containing 5% (v/v) probiotic lysate for an additional 24 h [18 (link)].

The transwell migration of human monocytes (PromoCell, USA) was studied in 24-well cell culture plates with polycarbonate membrane inserts having pores size of 5 μm (Corning, USA). The media used were phenol red free RPMI 1640 (Gibco, USA). The upper chamber of all wells contained a final volume of 200 μL medium containing 0.5% BSA, Sigma, USA, wherein 1 × 10⁵ cells were suspended. The lower chambers of all wells contained a final volume of 600 μL medium containing 10 ng/mL of monocyte chemoattractant protein-1 (MCP-1) (Abcam, USA) (except negative controls which contained only medium). Increasing concentrations (0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, and 1000 ng/mL) of recombinant phosphorylated HSP27 (Enzo Life Sciences) were placed in either upper or lower chambers in separate experiments. We also compared phosphorylated HSP27 with receptor activator of NF_kB ligand (RANKL), an osteoclast factor known to attract monocytes. For this, recombinant human RANKL (100 ng/mL, Invitrogen, USA) was placed in lower chambers and phosphorylated HSP27 (10 ng/mL) was placed in either upper or lower chambers. Migration was allowed to proceed for two and half hours at 37°C in 5% CO₂. The migrated cells suspended in the lower chamber were counted using a haemocytometer. All assays were performed thrice in triplicate.