Facs celesta flow cytometry system

Cells at a density of 1 × 10⁶ cells/mL were seeded in a 10-cm (bEnd.3) or 6-cm (MBECs) petri dish and cultured overnight. Various conditions of NAC were used to treat cells as mentioned in “Bacterial infection and antioxidant treatment” section. P. gingivalis was allowed to infect cells for 90 min before replaced with fresh medium and cultured for a further 24 h. Cells were enzymatically collected and proceeded with FITC Annexin V apoptosis detection kit (Cat. # 556547; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Briefly, cells were resuspended with 1XAnnexin V binding buffer. Then, 100 μL of the solution was transferred to 5 mL tube. Five microliters of FITC Annexin V and/or PI were added to the solution and incubated for 15 min in the dark. After the incubation period, 400 μL of 1XAnnexin V binding buffer was added to the solution and analyzed with BD FACSCanto™ II flow cytometry or BD FACSCelesta™ flow cytometry system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Unstained cells, cells stained with FITC Annexin V (no PI), and cells stained with PI (no FITC Annexin V) were used to set up compensation and quadrant before acquiring results in each experiment. Cell population of quadrant 2 (FITC Annexin V +/PI −) and quadrant 4 (FITC Annexin V +/PI +) were quantitated and calculated as a percentage of apoptotic cell.

The production of intracellular reactive oxygen species (ROS) by bEnd.3 and MBECs after P. gingivalis infection was measured by the conversion of DCFH-DA, a cell-permeable non-fluorescent probe, into highly fluorescent DCF upon oxidation as described previously [35 (link),55 (link)]. The bEnd.3 or MBECs at a density of 5 × 10⁵ cells/mL were cultured overnight in a T25 flask. Different concentrations of NAC were incubated with cells. After incubation period, NAC was removed. Cells were incubated with 50 μM of DCFH-DA (Cat. # D6883; Sigma-Aldrich, St. Louis, MO, USA) for 30 min followed by incubation with 500 μM of hydrogen peroxide solution (H₂O₂; Cat. # 18304; Sigma-Aldrich, St. Louis, MO, USA) or P. gingivalis for 90 min. Cells were collected enzymatically and analyzed with BD FACSCanto™ II flow cytometry or BD FACSCelesta™ flow cytometry system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Human pancreatic islets of non-diabetic subjects were cultured with standard medium, or in the presence/absence of IGFBP3 and/or in the presence/absence of Ecto-TMEM219 for 72 h as previously described (see “Pancreatic islets and Interventional studies”). Islets were detached with Versene (ThermoFisher Scientific) and stained with the BD Horizon™ Fixable Viability Stain 510 (FVS510, BD Biosciences 564406, San Jose, CA), which ensures accurate assessment of cell viability in samples after fixation and/or permeabilization as per manufacturer’s instructions. After being stained with BD Horizon™ Fixable Viability Stain 510, cells were next fixed and permeabilized with Fixation and Permeabilization Solution Kit (554714, BD Biosciences, San Jose, CA)^{51 (link)} and finally stained with guinea pig anti-insulin antibody (1:200, Guinea Pig, ThermoFisher Scientific, PA1-26938) followed by AlexaFluor488 anti-guinea pig (1:200, ThermoFisher Scientific, A-11073). Flow cytometry analysis was performed using a BD FACS Celesta flow cytometry system (BD Biosciences) and analyzed using Flowjo software (Version 6 and Version 10, Tree Star, Ashland, OR).