6 well plate

HEK cells were plated at 70% confluence in a 6-well plate (VWR) and transfected using Lipofectamine 2000 (Invitrogen) per the manufacturer’s directions. After 24 h, cells were injured with 30 µM ionomycin and cell scrappers, then they were pelleted at 300 g for 5 min and the cell pellet was solubilized in RIPA buffer (Life technologies) and protease inhibitor cocktail (Life technologies). Samples were separated by SDS-PAGE on 3–8% NuPAGE Tris-Acetate gels (Life technologies) using Chameleon Duo as a size marker and transferred onto nitrocellulose membranes (at 100 V for 3 h at 4°C). Membranes were blocked using fluorescent WB blocking buffer (Tebu-bio) in TBS 1X for 1 h at room temperature. Primary antibodies (Hamlet-1, 1:150, Abcam 75571) were then diluted in blocking buffer and incubated overnight at 4°C. After washing in TBS-T, membranes were then incubated with secondary antibodies (IRDye 800CW Donkey anti-mouse, Li-Cor, ref 926-32212), which were diluted 1:10000 in blocking buffer for 45 min at room temperature. The membranes were washed in TBS-T and developed using NIR Fluorescence LI-COR. Actin (1:5,000, Merck MA1501r) was detected using a dilution of 1:10000 of secondary antibodies (IRDye 680RD Donkey anti-mouse, Li-Cor, ref 925-68072).

ARPE-19 cells were seeded in a 6-well plate (VWR, Mississauga, ON, Canada. cat # 82050-842) at 600,000 cells/well (60,000 cells/cm²) and reached confluency at ~120,000 cells/cm². Cultures were maintained for up to 70 days in 2 mL of ARPE-19 culture media that consisted of: DMEM/F-12, HEPES (Thermo Fisher Scientific, Mississauga, ON, Canada. cat # 11330057), 10% FBS (VWR. cat # 97068-085), and 1% Pen/strip (Thermo Fisher cat # 15140122). Media was changed every 48 h by replacing the entire old media volume with 2mL of fresh media. For all experiments, E-RPE cells were cultured in an identical manner to ARPE-19 cells, using ES-RPE culture media that consisted of: 70% DMEM; 30% F12; 2% B-27 supplement and 1% Pen/strip (Thermo Fisher. cat # 11965, 11765, 17504, and 15140122).
To assess the oxygenation status of the cells under the outlined culture conditions, oxygen delivery was calculated using our previously published method [66 (link)]. A RPE oxygen consumption rate of 42 amol∙cell⁻¹∙s⁻¹ was used for the calculation [67 (link)]. We determined that cultured RPE cells should be receiving an adequate amount of oxygen, with a local oxygen concentration of 1.42 × 10⁻⁴ mol/L at the cells and a maximum oxygen delivery rate of 191 amol∙cell⁻¹∙s⁻¹.

Overnight cultures (18 hours of growth in a 37 °C incubator, shaking at ~90 rpm) of S. aureus, A. baumannii, and P. aeruginosa were diluted to an OD₆₀₀ of 0.5. One milliliter of a diluted culture was dispensed to each well in a 6-well plate (VWR Intnl Inc, 29442–042) and left for 1 hour for cell attachment. The wells were then washed twice with 1 mL of sterile TSB. TSB (2 mL) was added, and the plates were incubated for 24 hours at room temperature. The medium was replaced, and incubation was continued for 48 hours total. LB was used for P. aeruginosa biofilm growth instead of TSB.