12 well culture plate

The myoblast cell line C2C12 (European Collection of Cell Cultures, ECACC) was used as a muscle cell model, as described by Orellana et al.^{32 (link)}. Briefly, myoblasts were seeded onto the scaffolds, placed in a 12-well plate, at a density of 2.5 × 10⁵ cells/cm². As a control, we used commercial cell culture plastic. For both the scaffolds and control we used 12-well culture plates (Falcon, Germany, Cat. N°: AZ1035C353043). Myoblasts proliferated for 3 days under standard conditions (37 °C and 5%CO₂) using proliferation medium: DMEM high glucose with L-glutamine (2 mM) (Gibco, Life Technologies, USA) and 10% fetal bovine serum (Biologicals Industries, Israel). Then, cells were differentiated for 7 days using differentiation medium: DMEM high glucose with L-glutamine (2 mM) and 2% horse serum (Gibco, Life Technologies, USA). For more detail, see^{15 (link),32 (link),33 (link)}.

To measure the monolayer cell permeability, SCE cells were grown on polycarbonate membrane inserts (0.4 μm pore size and 12 mm diameter; Corning Transwell; Sigma-Aldrich) on 12-well culture plates (BD Falcon, Franklin Lakes, NJ, USA) in DMEM supplemented with 10% FBS at 37 °C in 5% CO₂ until confluent as previously described⁶⁰ . The volume of the applied medium was 0.5 mL on the apical side (inside of the membrane inserts) and 1.5 mL on the basal side (outside of the membrane inserts). Two weeks after seeding, SCE-cell monolayers were exposed to the conditioned medium, and a 4 kDa FITC-dextran dye (Sigma-Aldrich) was simultaneously applied at 50 μM to the basal compartment of the wells. The medium was collected from the apical side for fluorescence measurements at 3, 6, and 24 h after adding the dye, and the same volume of fresh culture medium was replaced. The concentration of FITC-dextran in the collected medium was measured using a multimode plate reader (Multi Microplate Reader, MTP-800AFC; Corona Electric, Ibaragi, Japan), with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Fluorescence intensity of the normal medium was measured as the background concentration in each experiment, and each experiment was repeated at least three times.

Hp strains from frozen stocks were cultured on Brucella agar plates supplemented with 7% laked horse blood (Oxoid, Hampshire, UK) and incubated for 3 days at 37 °C under microaerophilic conditions in accordance with previously published protocols [18 (link)]. A sterile cotton swab was used to harvest bacteria after incubation. Bacteria were then suspended in brain heart infusion (BHI) broth (Oxoid, Hampshire, UK) additioned with 0.5% β-cyclodextrin (Sigma-Aldrich, St. Louis, MO, USA) and 0.4% yeast extract (Oxoid, Hampshire, UK). Aliquots of 2 mL of this suspension, adjusted to 5 × 10⁶ CFU/mL concentration, were inoculated in the wells of 12-well culture plates (BD Falcon, Franklin Lakes, NJ, USA), where sterilized round glass coverslips were placed vertically in order to promote the adhesion of Hp cells at the air-liquid interface. BHI broth (without bacteria) was the negative control. Plates were incubated for 4 days at 37 °C under microaerophilic conditions. Biofilms grown on the glass coverslips were washed twice with phosphate-buffered saline (PBS, Gibco, Life Technologies, Carlsbad, CA, USA) to remove excess bacterial cells and immediately set for imaging at room temperature.

A bacterial Adhesion assay was performed to confirm adherence by visualization of the adhered bacteria on infected GMECs [46 ]. About 1.5 × 10⁵ GMECs were seeded in 12 well culture plates (BD Falcon). At 80% confluency, the growth medium was replaced by an infection medium (Medium with bacteria). Monolayers/wells were infected with hk bacteria (10⁶ cfu/ml) at an MOI of 1:100 for 3 h at 37 °C with 5% CO₂. Uninfected control wells with infection media with no bacteria were also included. The wells were stained with 20% Giemsa stain (Sigma-Aldrich) for 30 min and visualized under Inverted Microscope (IX73, Olympus, Japan) at 100×.

MMP (ΔΨm) levels were measured using rhodamine 123 (Sigma-Aldrich Co.; Ex/Em = 485 nm/535 nm) and JC-1 dyes (Enzo Life Sciences ; Ex/Em = 515 nm/529 nm). Briefly, 5 × 10⁴ cells in 12 well culture plate (BD Falcon) were incubated with the indicated concentration of SAHA for 24 hours. Cells were washed twice with PBS and incubated with 0.1 μg/ml rhodamine 123 or 10 μg/ml JC-1 at 37^°C for 30 min. For staining nucleus, cells were incubated with 500 nM 4′, 6′-diamidino-2-phenylindole (DAPI, Life Technologies, Ex/Em = 358 nm/461 nm) at 37^°C for 30 min. The intensity of rhodamine 123 staining was determined by Accuri C6 flow cytometry (BD Sciences). The negative staining of rhodamine 123 from cells indicated the loss of MMP (ΔΨm) in cells. After incubation with JC-1 and DAPI, cells were washed three times with PBS and images were collected by using a fluorescence microscope (FLoid^® Cell Imaging Station, Life Technologies) in × 400 magnification. Green fluorescence indicates a monomer at low ΔΨm and red fluorescence presents high ΔΨm.

Surface expression of TNFR1 was detected by PE conjugated TNFR1 antibody (Santa Cruz Biotechnology). PE conjugated Armenian hamster IgG (Santa Cruz Biotechnology) was used as an isotype control. Briefly, 5 × 10⁴ cells in 12 well culture plate (BD Falcon) were incubated with the indicated 5 μM SAHA for 24 hours. Then cells were washed twice with cold PBS and added 500 μl of PBS at a concentration of 1 × 10⁶ cells/ml. Ten microliters of PE conjugated TNFR1 antibodies or five microliters of PE conjugated Armentian hamster IgG were added to these cells, which were analyzed with an Accuri C6 flow cytometer (BD Sciences).