human vaginal epithelial cell growth media with serum  (Celprogen Inc)

Journal: bioRxiv

Article Title: Bacteriophage-mediated reduction of uropathogenic E. coli from the urogenital epithelium

doi: 10.1101/2025.10.06.680661

Figure Lengend Snippet: Human vaginal epithelial VK2 cell monolayers were pretreated with ΦHP3 and ΦCocktail (10 8 PFU) for 1 hour and non-adherent phage was removed by washing (Adherent Φ) or unwashed to retain all phage (Total Φ). ( A ) ΦHP3 PFU recovered from filtered supernatant (cell-free) or lysed cells (cell-associated) for Adherent Φ and Total Φ conditions. Monolayers were infected with 10 6 CFU of UPEC for 30 min followed by cell lysis and plating to quantify adherent bacteria. ( B ) Adherence of phage-sensitive WT UTI89 to ΦHP3 and ΦCocktail pretreated cells under Adherent Φ and Total Φ conditions. ( C ) Adherence of phage-resistant UTI89 Φ R to ΦHP3 pretreated cells under Adherent Φ and Total Φ conditions. For confocal microscopy, VK2 monolayers were infected with 10 6 CFU UTI89-RFP (red) for 3 hours, prior to treatment with 10 8 PFU SYBR-Gold labeled ΦHP3 (green) with image acquisition immediately after phage addition. ( D ) Representative images with cell nuclei stained with Hoechst (blue) and cell membranes visualized with wheat germ agglutinin (magenta). Arrows indicate phage co-localization with host cells (green), UPEC co-localization with host cells (red), or phage co-localization with UPEC (yellow). Experiments were performed in at least technical duplicate across four to eight independent replicates (A-C) or twice independently (D). Points represent medians of experimental technical replicates and lines represent medians with interquartile ranges (A-C). Data were analyzed by two-way ANOVA with uncorrected Fisher’s LSD (A), two-way ANOVA with Holm-Šídák’s multiple comparisons test (B), or Kruskal-Wallis with Dunn’s multiple comparisons test (C), ** p <0.01, **** p <0.0001.

Article Snippet: Immortalized human vaginal epithelial cells (VK2/E6E7, ATCC CRL-2616) were grown in keratinocyte serum-free medium (KSFM) containing 50 mg/mL bovine pituitary extract and 0.1 ng/mL human recombinant epithelial growth factor.

Techniques: Infection, Lysis, Bacteria, Confocal Microscopy, Labeling, Staining

Journal: bioRxiv

Article Title: Bacteriophage-mediated reduction of uropathogenic E. coli from the urogenital epithelium

doi: 10.1101/2025.10.06.680661

Figure Lengend Snippet: For invasion assays, human vaginal epithelial VK2 cell and bladder carcinoma HTB-9 cell monolayers were pretreated with ΦHP3 and ΦCocktail (10 8 PFU) for 1 hour and non-adherent phage was removed by washing (Adherent Φ) or unwashed to retain all phage (Total Φ). Monolayers were infected with 10 6 CFU of UPEC for 2 h, gentamicin treatment for 1 h to kill extracellular bacteria, followed by cell lysis and plating to quantify invasive bacteria. VK2 cell invasion of phage-sensitive WT UTI89 to ΦHP3 and ΦCocktail pretreated cells ( A ), or UTI89 Φ R to ΦHP3 pretreated cells ( B ), under Adherent Φ and Total Φ conditions. HTB-9 cell invasion of phage-sensitive WT UTI89 to ΦHP3 and ΦCocktail pretreated cells ( C ), or UTI89 Φ R to ΦHP3 pretreated cells ( D ), under Adherent Φ and Total Φ conditions. For intracellular survival assays, VK2 and HTB-9 monolayers were infected with 10 6 CFU of UPEC for 2 h, gentamicin treatment for 1 h to kill extracellular bacteria. Monolayers were then treated with 10 8 PFU phage and gentamicin for 24 h, followed by cell lysis and plating to quantify intracellular bacteria. VK2 intracellular UTI89 or UTI89 Φ R after treatment with ΦHP3 ( E ), or UTI89 after treatment with ΦCocktail ( F ). HTB-9 intracellular UTI89 or UTI89 Φ R after treatment with ΦHP3 ( G ), or UTI89 after treatment with ΦCocktail ( H ). ( I ) ΦHP3 adherence to VK2 and HTB-9 cells after 30 min of incubation with 10 8 PFU, followed by washing and cell lysis. ( J ) IL-8 release in VK2 cell supernatant after 6 h of infection with 10 6 CFU UTI89 with or without 10 8 PFU ΦHP3 as measure by ELISA. Experiments were performed in at least technical duplicate across three to twelve independent replicates. Points represent medians of experimental technical replicates and lines represent medians with interquartile ranges (A-J). Data were analyzed by two-way ANOVA with Holm-Šídák’s multiple comparisons test (A,C,E,G), Kruskal-Wallis with Dunn’s multiple comparisons test (B,D), paired t test (F, H), Mann-Whitney U test (I), or two-way ANOVA with uncorrected Fisher’s LSD (J), * p <0.05, *** p <0.001.

Article Snippet: Immortalized human vaginal epithelial cells (VK2/E6E7, ATCC CRL-2616) were grown in keratinocyte serum-free medium (KSFM) containing 50 mg/mL bovine pituitary extract and 0.1 ng/mL human recombinant epithelial growth factor.

Techniques: Infection, Bacteria, Lysis, Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY