Cptio

The ECS, the pbECS, and further variations in solutions used in Ca²⁺ imaging experiments were freshly prepared, and Table 1 lists the components. The Ponceaus S solution was composed of 0.2% Ponceau S, 3% trichloroacedic acid, and 3% sulfosalicylic acid. Sources for chemicals and solutions: Dulbecco’s phosphate buffered saline (DPBS) (Sigma-Aldrich, Steinheim, Germany), fura-2 AM and pluronic F-127 (Biotium, Fremont, CA, USA), ionomycin (2 µM, Enzo Life Sciences, Farmingdale, NY, USA), DMSO (Sigma-Aldrich), histidine (3 µM, Sigma-Aldrich), NaN₃ (Sigma-Aldrich), 30% (w/w) H₂O₂ (6 µM, Sigma-Aldrich), cPTIO (250 µM, Cayman Chemical, Ann Arbor, MI, US), anti-nitrotyrosine antibody (MERCK, Darmstadt, Germany, 06-284), and alkaline phosphatase-conjugated rabbit secondary antibody (Cell Signaling, Danvers, MA, USA, 7054S). Dihydrorhodamine 123 (DHR 123) and the Fluorimetic Hydrogen Peroxide Assay Kit both from Sigma-Aldrich, 4,5-diaminofluorescein (DAF-2) from Enzo Life Sciences (Farmingdale, NY, USA) and the Singlet Oxygen Sensor Green Reagent purchased from Molecular Probes (Eugene, OR, USA) were used to perform the fluorescence spectroscopic analyses of CAP-induced reactive species.

4-Amino-5-methylamino-2′,7′-difluorescein (DAF-FM) was purchased from Invitrogen (Carlsbad CA, USA), and cPTIO was from Cayman Chemical (Ann Arbor, MI, USA). All other reagents, including quinine HCl, were obtained from Sigma-Aldrich unless otherwise indicated. Stock solutions of DAF-FM and cPTIO were made at 1:1,000 dilution in DMSO and were made fresh daily. All air–liquid interface (ALI) experiments were performed with Dulbecco’s PBS (1.8 mM Ca²⁺) on the apical surface of the cultures, while the basolateral side was bathed in a modified HEPES-buffered Hank’s Balanced Salt Solution (HBSS) with 1× MEM amino acids to provide a source of arginine (0.6 mM) for the production of NO.

White leghorn (WH) eggs were obtained from Poultry Research centre, Nandanam, Chennai. They were incubated at 38 °C in a humidified incubator as described previously [36 (link)], windowed and staged according to Hamburger and Hamilton (HH stage) [37 (link)]. The NO donors were dissolved in sterile water at mentioned concentrations. A small hole was made in the broad end of the day 0 egg with a sterile needle. 10 µL of NO donor, DEAN (100, 500 and 1000 µM in 1× PBS) or other NO donors (SNP, DETA) or quencher (cPTIO) were injected through the hole in the air sac using a sterile tip as a single dose at HH stages 0- HH stage 13 into the air sac. The eggs were then sealed with a sterile mediplast tape and incubated further from day 0 to day 12 depending on the experiment requirement. The control eggs were treated with 1X PBS or left without an injury in the egg shell. SNP and DEAN were obtained from Sigma-Aldrich, St. Louis while DETA, cPTIO were obtained from Cayman chemicals, Michigan. The authors declare that the research was conducted with prior approval from Institutional Biosafety and Ethical Committee (IBEC) of the AU-KBC Research Centre as per Annexure I; Project 02 (14-11-2011).