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. 2023 May 31;14:1138539. doi: 10.3389/fimmu.2023.1138539

Figure 6.

Figure 6

Flow cytometry analysis demonstrates an increased H3K27 acetylation in β-glucan-trained cells upon a stimulation by S. aureus. Osteoblasts-like MG-63 cells (20,000 cells/well) were seeded in wells of 24-well plates. Afterwards MG-63 cells were exposed to 100 µg/mL of β-glucan or to the cultivation medium for 24 h, followed by a 5-day incubation prior to infection with S. aureus (MOI 60:1) for 100 minutes, and then incubated for an additional 24 h as described in Material and Methods. As a positive control for histone acetylation an incubation of cells with 10 nM of TSA for 24 h were performed. At the end of the incubation, adherent cells were dissociated into single-cell suspension using Trypsin/EDTA treatment. They were then fixed in 4% paraformaldehyde/PBS for 20 min and permeabilized in 0.3% TritonX100/PBS for 10 min. Cells were subsequently incubated with anti-H3K27ac antibody (# 15562, Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, PE Conjugate) diluted in PBS/2%BSA (1:50) for 1 h. An isotype control antibody (#5742, Rabbit (DA1E) mAb IgG XP® Isotype Control, PE Conjugate) was used to exclude nonspecific binding. Afterwards, cells were analyzed for H3K27 acetylation using an Accuri C6 flow cytometer (FL2 channel). Data were collected from 20,000 cells and analyzed using the CFlow software (Becton Dickinson). Values of the respective mean fluorescence intensities (MFIs) were compared to that of the control. Black graph corresponds to the isotype control. (A) Double arrow shows the shift between the fluorescence corresponding to the level of acetylation in control cells (represented by the green line) and TSA-treated cells (red line). (B) Double arrow shows the shift between the fluorescence corresponding to the level of acetylation in control cells (green line) and in S. aureus-infected cells (red line). Dotted double arrow shows the shift between the fluorescence corresponding to the level of acetylation in control cells (green line) and β-glucan pretreated cells exposed to S. aureus (blue line). Two independent experiments in triplicate were performed. The results of the one representative experiment is shown.