Investigating PM10 and P. aeruginosa Effects on HCET Cells

HCET cells (HCE-2 [50.B1], ATCC, Gaithersburg, MA) were cultured in Keratinocyte-serum free (KSF) medium (Gibco, Grand Island, NY) with 5ng/ml human recombinant EGF, 0.05mg/ml bovine pituitary extract, 0.005mg/ml insulin, and 500ng/ml hydrocortisone as previously described (Somayajulu et al., 2023 (link)). Cells were treated with PM₁₀ (0, 25, 50, 100, 200, 500, 800 and 1200µg/ml for 24h for the MTT assay. For all other experiments, cells were incubated with 100μg/ml (per cell viability data from dose curve; 75-80% viability) PM₁₀ at 37°C and 5% CO₂ for 24hr. To investigate the combined effects of PM₁₀ on P. aeruginosa infected cells, a subset of cells were challenged with strain 19660 at a multiplicity of infection (MOI) of 20 for 3h. To assess the effects of SKQ1 (BOC Sciences, Shirley, NY, USA), another subset of cells were incubated with 50nM SKQ1, 1h before PM₁₀ exposure (Somayajulu et al., 2023 (link)) and then challenged with strain19660 at similar MOI. Another group of HCET were challenged with strain 19660 at a MOI of 20 for 3h to assess the effects of bacteria alone on these cells. Phase contrast microscopy was used to photograph cell preparations using a Leica EL 6000 microscope (Deerfield, IL, USA). All the images were acquired at the same magnification and processed similarly.