12 well culture plate

A nuclei-containing fragment of the expanded gel was incubated with primary rabbit antibodies against the centromeric H3 variant CENH3 of barley (Houben et al. 2007 (link)) diluted 1:1000 in 400 μl of antibody solution (2.5% BSA, 0.05% Triton X-100, 1×PBS) in a 12-well culture plate (Greiner) and incubated overnight for at least 15 h at RT. Next, the primary antibody solution was removed, and the gel was re-expanded in distilled water until it reached the size before antibody incubation. Afterwards, the gel was incubated with secondary anti-rabbit Alexa488 antibodies (1:200, no. 711-545-152, Jackson ImmunoResearch) in 400 μl antibody solution and incubated at 37°C for 1 h followed by 2 h at RT. Additionally, 3–5 μg/ml DAPI solution can be added together with the secondary antibodies to enable chromatin staining. Finally, the gel was re-expanded in distilled water and subjected to microscopic observation in a 22×22-mm coverslip chamber “Chamlide” (Live Cell Instruments, catalogue no. CM-S22-1). The simplified schema of these protocol steps is shown in Fig. 1b.

AGS cells or primary human gastric cells were seeded in a 12-well culture plate (Greiner, Frickenhausen, Germany) at a density of 1.5 × 10⁴ cells per cm² and left for 24 hours as previously described [27 (link)]. After which, cells were treated with H. pylori WT or isogenic mutants for an additional 24 hours. Where indicated, rGGT (10mU), serine-borate complex (SBC), a competitive GGT inhibitor [28 (link)] (2-10mM; Sigma-Aldrich, St Louis, MO) or ammonium chloride (1-3mM) was incubated with AGS cells. At the respective time-points, cells were inspected for vacuolation using phase contrast microscopy. Extent of vacuolation was then quantified using neutral red uptake assay as previously described with slight modifications [16 (link)]. Briefly, the medium overlaying the cells was removed and replaced with 0.2 ml of freshly prepared 0.05% neutral red in PBS containing 0.3% bovine serum albumin (BSA) for 5 min. Cells were then washed thrice with 0.5 ml of PBS/0.3% BSA. The neutral red dye taken up by the vacuoles was then extracted from the cells by the addition of 0.2 ml of acidified ethanol (70% ethanol, 0.37% HCl). The optical density was measured with a microplate reader (BioRad Laboratories, Richmond, VA) at 534 nm with subtraction of absorbance at 405 nm. Neutral red dye uptake is expressed as absorbance per 10⁵ cells. All experiments were done in triplicates.

The maximum non-toxic dose of recombinant S100A8, S100A9 and combined S100A8/S100A9 for primary conjunctiva fibroblasts treatment were pre-determined to be 40, 25 and 20 µg/ml respectively. Primary conjunctiva fibroblast cells were seeded (200,000 per well) in a 12-well culture plate (Greiner bio-one) and cultured in serum-free DMEM/F12 medium (Gibco) for 24 hrs. Cells were washed with PBS and treated with S100A8, S100A9 and S100A8/A9 proteins in fresh media for 6 or 24 hrs. Control cells were given fresh medium without recombinant protein.