ATP Quantification in Frozen Lung Tissue

The ATP content was quantified from frozen whole lung tissue using ATP Assay Kit (Biovision, Inc. Milpitas, CA), following manufacturer’s instructions and previously described protocol [64 (link)]. Briefly, frozen lung tissues were pulverized at − 80 °C followed by homogenization using assay buffer provided by manufacturer. Total Protein was quantified using BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific, Waltham, MA). The standard curve for ATP was generated over a range of 20 nM to 1 nM. The phosphorylated glycerol from lung homogenates was measured at 570 nm using SpectraMax®iD5 (Molecular Devices, CA) plate reader after incubation with the probe for approximately 90 min. All results were normalized to total protein measured using BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific, Waltham, MA) for quantification.