P lck

Lungs were isolated and chopped into very small pieces using fine surgical scissors followed by incubation in digestion cocktail containing collagenase and DNAse I in RPMI-1640. This led to formation of single cell suspension of pulmonary cells which was then utilized for flow experiment as done earlier [22 (link)]. Single cells from pulmonary tissue were first incubated with monoclonal antibodies against cell surface antigen, i.e., CD4 (BioLegend, San Diego, CA, USA) fluorescently conjugated to APC-Cy7/APC/FITC. After the normal step of fixation and permeabilization (Miltenyi Biotech, Bergisch Gladbach, North Rhine-Westphalia, Germany) of pulmonary cells in the suspension, cells were then labeled with monoclonal antibodies against intracellular proteins, such as p-LCK, p-ITK, p-PLCγ, NFATc1, p-NFkB, IL-4, IL-5, IL-10, Foxp3, GATA-3, iNOS, and Nitrotyrosine (R&D Systems, Minneapolis, MN, USA; BioLegend, San Diego, CA, USA). Immunolabeled leukocytes in pulmonary cell suspension were then run and 10,000 events were acquired on a flow cytometer (Beckman Coulter, Indianapolis, IN, USA), followed by evaluation of fluorescently conjugated intracellular/cell surface proteins using Cytomics FC 500 software as stated before [22 (link)].