Human bronchial epithelial 16HBE14 cells (Gruenert et al., 1988 (link)), kindly provided by Dr. Kirsten Spann (Queensland University of Technology), were seeded in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (sMEM) at an approximate density of 2 ∗ 105 cells/ml into 24-well culture dishes (Greiner Bio-One, Kremsmünster, Austria) (1 ml/well) for adherence and invasion assays or into 175-cm2 flasks (Corning, Tewksbury, MA, United States) (40 ml/flask) for maintenance. Confluent 16HBE14 monolayers were washed once with prewarmed sMEM and then infected with Hi2019WT, Hi2019ΔdmsA, or Hi2019ΔdmsA_c diluted in sMEM to 2 ∗ 107 bacteria/ml, giving a multiplicity of infection (MOI) of 1:100 (epithelial cells: bacteria). Bacterial adherence and invasion were determined as described previously (Dhouib et al., 2015 (link), 2016 (link)). Determination of intracellular bacteria used a standard gentamicin-protection assay (St Geme and Falkow, 1990 (link)). In brief, infected cells were washed to removed planktonic and loosely adherent bacteria and incubated for 1 h in sMEM containing 50 μg/ml gentamicin followed by saponin lysis and serial dilution for determination of bacterial CFU present. Assays used at least three biological replicates; replicate experiments were carried out on different days.
24 well culture dishes
Manufactured by Greiner
Sourced in Austria, Germany
The 24-well culture dishes are a type of laboratory equipment used for cell culture experiments. They provide a standardized platform with 24 individual wells for growing and maintaining cells in a controlled environment.
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3 protocols using 24 well culture dishes
1
Quantification of Bacterial Adherence and Invasion in Human Bronchial Epithelial Cells
2
Luteal Cell Expression Dynamics
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The dispersed luteal cells were seeded at 2.0 × 105 viable cells per milliliter in 24-well culture dishes (no. 662160; Greiner Bio-One, Frickenhausen, Germany) to evaluate mRNA expression. The cultures were kept in a humidified atmosphere of 5% CO2 in air at 38 C in an N2–O2–CO2-regulated incubator (no. BNP-110; ESPEC, Osaka, Japan). After 24 h of culture, the culture medium was replaced with fresh medium containing 0.1% BSA (no. 15408; Roche Diagnostics, Mannheim, Germany); 5 ng/ml sodium selenite (no. S5261; Sigma-Aldrich); 5 µg/ml holo-transferrin (no. T3400; Sigma-Aldrich); and 0.01, 0.1 or 1 μM PGF (no. 16010; Cayman chemical, Ann Arbor, MI, USA), 0.05, 0.5 or 5 nM TNF (Dainippon Pharmaceutical, Osaka, Japan) or 0.05, 0.5 or 0.5 nM IFNG (Kindly donated by Dr S Inumaru, NIAH, Ibaraki, Japan) for 24 h. The concentrations of PGF, IFNG and TNF for combination treatment were determined based on those that had the most
effect on the expression of MMP mRNA in single treatments with PGF, IFNG and TNF. The luteal cells were also exposed to 1 μM PGF in combination with 5 nM IFNG or 5 nM TNF, 5 nM IFNG in combination with 5 nM TNF, 1 μM PGF in combination with 5 nM IFNG and 5 nM TNF for 24 h.
effect on the expression of MMP mRNA in single treatments with PGF, IFNG and TNF. The luteal cells were also exposed to 1 μM PGF in combination with 5 nM IFNG or 5 nM TNF, 5 nM IFNG in combination with 5 nM TNF, 1 μM PGF in combination with 5 nM IFNG and 5 nM TNF for 24 h.
3
Propagation of Human Bronchial Epithelial Cells
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Human bronchial epithelial 16HBE14 cells (Gruenert et al., 1988 (link)), kindly provided by Dr Kirsten Spann (Queensland University of Technology), were propagated in MEM - GlutaMAX™ (Gibco®, Life Technologies), supplemented with 10% fetal calf serum (Gibco®, Life Technologies, Cat.-No. 16000-044; sMEM). The cells were seeded into individual wells of 24-well culture dishes (Greiner Bio-One, Cat.-No. 662160) at an approximate density of 2*105 cells/well, incubated at 37°C with 5% CO2 until reaching confluence, then used for infection studies.
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