One day after the end of sensitization, animals were sacrificed by i.p. administration of 50 mg/kg ketamine (CAS Number: 1867-66-9, Sigma Chemical Ltd., UK) and 5 mg/kg xylazine (CAS Number: 23076–35-9, Sigma Chemical Ltd., UK). The left lung was washed with one mL saline five times (5 mL totally). BALF was centrifuged at 2500 g at 4 °C for 10 min and supernatant was stored at − 70 °C [28 (link)] until analysis.
Cytokine and inflammatory levels including interleukin 4 (IL-4, Cat Number: ab100770, Abcam Company, Cambridge, MA, USA), interferon gamma (IFN-γ, Cat Number: ab46107, Abcam Company, Cambridge, MA, USA), immunoglobulin E (IgE, Cat Number: ab157736, Abcam Company, Cambridge, MA, USA), phospholipase A2 (PLA2, Cat Number: MBS262388, MyBioSource Inc., San Diego, California, USA) and total protein (TP, Cat Number: 128500, Pars Azmoon. Co., Iran) in the BALF were measured by enzyme-linked immunosorbent assay (ELISA) sandwich method with appropriate protocol recommended by company. The ratio of IFN-γ/IL4 as an index of Th1/Th2 was also calculated.
Cytokine and inflammatory levels including interleukin 4 (IL-4, Cat Number: ab100770, Abcam Company, Cambridge, MA, USA), interferon gamma (IFN-γ, Cat Number: ab46107, Abcam Company, Cambridge, MA, USA), immunoglobulin E (IgE, Cat Number: ab157736, Abcam Company, Cambridge, MA, USA), phospholipase A2 (PLA2, Cat Number: MBS262388, MyBioSource Inc., San Diego, California, USA) and total protein (TP, Cat Number: 128500, Pars Azmoon. Co., Iran) in the BALF were measured by enzyme-linked immunosorbent assay (ELISA) sandwich method with appropriate protocol recommended by company. The ratio of IFN-γ/IL4 as an index of Th1/Th2 was also calculated.
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