Sf9 Cell Line Irradiation Protocol

Sf9 cell line, (originally established from the ovaries of Spodoptera frugiperda) obtained from National Institute of Immunology, New Delhi, India, was maintained as semi-adherent monolayer in the 25 cm² culture flasks (Falcon, BD Biosciences, USA) at 28 °C in Grace’s insect cell medium (Sigma, St Louis, MO, USA) supplemented with 3.33 g/l lactalbumin hydrolysate (Sigma), 3.33 g/l yeastolate (BD Biosciences, San Jose, CA, USA), 0.35 g/l NaHCO3 and antibiotics (Penicillin sodium salt 50,000 U/l, streptomycin sulfate 50,000 μg/l, Nystatin 2000 μg/l from 500,000 USP U/mg; Sigma). Growth medium (pH 6.2) was prepared by adding 10% heat inactivated Fetal Bovine Serum (FBS) (Sigma, USA) and stored at 2–8 °C. Cells were regularly subcultured twice a week in exponential phase and were seeded at 10⁶ cells/flask (35,000–40,000 cells cm⁻²) in 5 ml of growth medium. Irradiation was carried out in the exponential phase of cell growth at a dose rate of 19.16 Gy min⁻¹ in Gamma Chamber-5000 (Board of Radiation and Isotope Technology, Department of Atomic Energy, Mumbai, India). Based on our previous studies^{19 (link),24 (link),48 (link),49 (link)} 24 h post-irradiation time point was selected (unless specified otherwise) for cell death and protein expression analysis, since Sf9 cells undergo substantial cell death primarily at 24h-48h following irradiation at high doses.