Accutase cell detachment solution

To prepare the heart tissues, excess fat was carefully removed using fine forceps, and the tissues were washed with cold 1× phosphate-buffered saline (pH 7.4) and placed in RPMI 1,640 with 10% fetal bovine serum and 1% penicillin-streptomycin. The heart tissues were then transferred to Accutase cell detachment solution (Corning, Corning, NY, United States) and cut into small pieces using fine scissors. After digestion for 60 min with gentle rocking at 37°C, the tissue samples were strained through a 70-µm cell strainer. The homogenates were washed twice with RPMI 1640 with 10% fetal bovine serum and 1% penicillin-streptomycin and centrifuged at 300 g for 5 min at 20°C. To remove erythrocytes, the cell pellets were resuspended in 1.0 mL red blood cell lysis buffer (BioLegend, San Diego, CA, United States), incubated for 10 min at room temperature, and diluted to 10 mL with RPMI 1640 with 10% fetal bovine serum and 1% penicillin-streptomycin. The samples were centrifuged at 300 g for 5 min at 20°C, and the resulting cell pellets were then resuspended in 0.5 mL commercial cell freezing media until use.

The HMEC-1 cultures in the plateau phase were subjected to gamma radiation at doses ranging from 0.1 Gy to 8 Gy, using a dose rate of 0.4 Gy/min, at the Armed Forces Radiobiology Research Institute (AFRRI) 60-Cobalt facility, as previously described [52 (link)]. Following radiation, the cells were detached using Accutase^® cell detachment solution (#25-058-CI, Corning, Corning, NY, USA), counted using the CountessTM II Automated Cell Counter (Catalog #, AMQAX1000, Invitrogen, Carlsbad, CA, USA), and re-plated in complete growth medium if the sub-culturing of cells in 6-well plates or 10-cm dishes was necessary. It should be noted that, to minimize DNA damage repair post-IR, the irradiated or sham-irradiated T25 flasks were temporarily placed on ice before cell harvest if they could not be handled immediately.

MDMs were plated at a density of 3x 10⁶/ml in 12-well plates (BD Falcon, 353043). Cysteamine was added 24h before infection. The cells were infected with B. cenocepacia at an MOI of 10. Viability assay was done by FACS analysis using the APC Annexin V apoptosis detection kit with Propidium Iodide (PI) (Biolegend 640932). The macrophages were detached by Accutase cell detachment solution (Corning 25058C1). Cells were collected, washed, and re-suspended in 100 μl of Annexin V Binding Buffer (Biolegend 422201), and then stained with 3 μl of Annexin V and 5 μl PI in the dark for 20 min at room temperature. The percentage of non-viable, apoptotic cells was assessed using flow cytometry (BD LSR 11 Flow Cytometer; BD Bioscience).