Trypsin edta solution

The cell cycle progression was determined by flow cytometer analysis [29] (link). Neuroblastoma cells were treated with SsnB or DMSO in DMEM with 10% FBS. Cells were collected after trypsinization with trypsin-EDTA solution (Atlanta Biologicals) and washed with 1× phosphate buffered saline (PBS). Approximately 1×10⁶ cells were suspended in 300 µl of ice-cold 1× PBS, fixed by adding 700 µl of chilled absolute ethanol (to make final ethanol concentration 70%) drop wise and incubated for overnight at −20°C. Cells were pelleted at 1000× rpm for 5 min at 4°C, washed with 1× PBS for 3 times and stained with propidium iodide (Sigma-Aldrich, St. Louis, MO) by adding 1 ml of propidium iodide staining solution (containing 20 µg/ml propidium iodide and 10 µg/ml DNase-free RNase A in 0.1% Triton X-100/PBS) for 1 h on ice in dark. Samples were subjected to fluorescence-activated cell sorting analyses utilizing Beckman Coulter flow cytometer (Beckman Coulter, Indianapolis, IN). A minimum of 10,000 events were analyzed in each experiment.

Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) medium, Roswell Park Memorial Institute (RPMI) 1640 medium, and insulin–transferrin–selenium (ITS) solution were purchased from Lonza (Walkersville, MD, USA). Antibiotics (penicillin–streptomycin solution), trypsin–EDTA solution, and fetal bovine serum (FBS) were from Atlanta Biologicals (Flowery Branch, GA, USA). Sodium pyruvate solution was purchased from Hyclone Laboratories Inc. (Logan, UT, USA). RNAzol RT was from Molecular Research Center (Cincinnati, OH, USA). The iScript™ complementary DNA (cDNA) Synthesis Kit and iTaq™ Universal SYBR Green Supermix were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The genes included TLR3, retinoic-acid-inducible protein 1 (RIG-I), IRF3, mitochondrial antiviral-signaling protein (MAVS), MDA5, NF-κB, interferon beta (IFNβ), IFNγ, porcine beta-defensin 2 (pBD2), and pBD3. The primers (Table S1 in Supplementary Material) were from Integrated DNA Technologies, Inc. (Coralville, IA, USA).