T25 cell culture flasks

A HaCaT keratinocyte cell line was cultured in high glucose DMEM supplemented with 2mM L-glutamine, 10% fetal bovine serum (FBS), 100U/ml penicillin and 100μg/ml streptomycin (Gibco, Life Technologies). For treatment, 1x10⁶ of HaCaT cells were plated in T25 cell culture flasks (Sarstedt, Germany) and incubated at 37°C with 5% CO₂ overnight. Before infection the cells were washed in phosphate-buffered saline (PBS) and suspended in the aforementioned medium without antibiotics.
Bacterial cultures were grown in MH broth at 37°C with shaking overnight. The cells (1x10⁸ CFU/ml) were harvested and washed in PBS. After centrifugation at 4500 x g for 5 min, the pellets were suspended in high glucose DMEM containing 2mM L-glutamine and 10% FBS.
Infection of HaCaT keratinocytes with A. baumannii was performed by addition of the bacterial cell suspension to a flask with HaCaT cells (multiplicity of infection was 100). The HaCaT and A. baumannii cells were coincubated at 37°C with 5% CO₂ for 21h. After treatment, the cell culture medium with bacterial cells was centrifuged at 405 x g for 5 min to pellet residual non-adherent HaCaT cells. For harvesting bacterial cells, supernatant was centrifuged at 4500 x g for 5 min and used for RNA isolation. Controls were incubated and purified as described above, but in the absence of HaCaT cells. Experiments were done in triplicate.

Approximately 1 × 10⁶ cells per flasks were seeded into T25 cell culture flasks (Sarstedt). After 24 h the culture flasks were filled up completely (air bubble-free) with media and were installed on the prewarmed RPM (37 °C, 5% CO₂). To investigate the ability of MCS formation, two RPM running time points were considered: media was completely removed from culture flasks after 24 h and after 48 h exposure to the RPM. Flasks were re-filled with fresh media for a further 24 h run on the RPM. After each run, cells were examined and photographed.

The human follicular thyroid carcinoma cell line FTC-133 was cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), and 1% penicillin/streptomycin (Life Technologies) at 37 °C and 5% CO₂ until use for the experiment. For RPM experiments FTC-133 cells were seeded at a density of 1 × 10⁶ cells per flask either in T25 cell culture flasks (Sarstedt, Nümbrecht, Germany) for mRNA and protein extraction or in slide flasks (Sarstedt) for immunofluorescence staining. Cells were given at least 24 h to attach to the bottom of the flasks.

HEK293T (DSMZ) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, PAN-Biotech, Aidenbach, Bavaria, Germany) supplemented with 10% fetal bovine serum (FBS, PAN-Biotech, Aidenbach, Bavaria, Germany) and 2 mM L-glutamine (PAN-Biotech, Aidenbach, Bavaria, Germany). The cells were maintained at 37 °C and 5% CO₂ adherently in T75 or T25 cell culture flasks (Sarstedt, Nümbrecht, North Rhine-Westaphalia, Germany) until the cells were 90% confluent. The cells were detached via trypsin/EDTA (Gibco, Life Technologies, Darmstadt, Hesse, Germany) and passaged every 3–4 days.

For AhR
knockdown experiments,
600,000 C3H10T1/2 cells were seeded in T25 cell culture flasks (Sarstedt,
#83.3910.002) and incubated for 24 h at 37 °C and 5% CO₂. Afterward, cells were bulk-transfected with 30 nM AhR siRNA (Dharmacon,
1:1 mixture of #J-044066-06-0005 and #J-044066-07-0005) or control
siRNA (Dharmacon, # D-001810-01-05) using the DharmaFECT1 transfection
reagent (Dharmacon, #T-2001-02) according to the instructions of the
manufacturer. After 24 h of incubation at 37 °C and 5% CO₂, transfected cells were trypsinized and seeded in 96-well
plates (6000 cells/well) for osteoblast differentiation assays or
in 12-well plates (60,000 cells/well) for RT-qPCR analysis. Cells
were incubated for 5 h at 37 °C and 5% CO₂ until they
were attached to the bottom of the wells and were then treated with
1.5 μM purmorphamine in the presence of DMSO or the compounds
for 48 or 96 h.

Knockdown of PLD1 expression was achieved by specific PLD1 small interfering RNA (siRNA; sc-44000; Santa Cruz Biotechnology, Heidelberg, Germany). Scrambled control RNA (scRNA; Qiagen GmbH, Hilden, Germany) was used for control experiments. MDA-NEO and MDA-HER2 cells (1 × 10⁶) were transfected with 100 nM siRNA and 100 nM scRNA, respectively, by electroporation using the Nucleofector™ 2b Device (Lonza, Cologne, Germany) as described in the user’s manual. Electroporated cells were seeded in T25 cell culture flasks (Sarstedt, Nümbrecht, Germany) for up to 72 h. Knockdown of PLD1 expression was verified by qPCR and PLD1 immunoprecipitation.