Interleukin 4 (il 4)

An ELISA was used to detect the release of TCIPA. The test samples were collected from the serum and lung tissue homogenate supernatants of three mice in each group. Mouse c–c motif chemokine ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1, catalog no. EK0568), mouse interleukin-1 alpha (IL-1α, catalog no. EK0391), mouse interleukin-1 beta (IL-1β, catalog no. EK0394), mouse macrophage-stimulating factor (M-CSF, catalog no. EK0445), mouse transforming growth factor-beta 1 (TGFβ1, catalog no. EK0515), mouse interferon-gamma (IFN-γ, catalog no. EK0375), mouse interleukin-4 (IL-4, catalog no. EK0405) and interleukin-10 (IL-10, catalog no. EK0417) ELISA kits were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). Mouse macrophage migration inhibitory factor (MIF, catalog no. H598-M-96, Nanjing, China) and mouse stromal cell-derived factor 1 (SDF-1, catalog no. H398-1-M-96, Nanjing, China) were purchased from Nanjing Jiancheng Bioengineering Institute.

The levels of specific IgE and IgG1 for HDM were determined by ELISA as described previously^{12 (link)}. Briefly, the ELISA microtiter plates were coated with HDM with at 1 ug/well in 100 µl carbonate buffered solution (CBS, 15 mM Na₂CO₃ and 35 mM NaHCO₃, pH9.5). After incubation (overnight, 4 °C), plates were washed 3 times with PBST (PBS containing 0.05% Tween 20), and blocked with 3% bovine serum albumin in PBS (3% BSA-PBS) (1 h, 37 °C). The serum (1:10 diluted with 3% BSA-PBS) or BSA (using as a negative control) were then added to each well and incubated (2 h, 37 °C). Subsequently, 100 µL of peroxidase-labeled goat anti-mouse IgE (1:2000) was added to each well. The plates were incubated (2 h, 37 °C). Following 3 washings, the reactions were developed with TMB (tetramethylbenzidine, 100uL/well) for 20 min and stopped by 50 µl 2 M H2SO4. The plates were read by an ELx808 absorbance microplate reader (BioTek, Shanghai, China) at 450 nm. The splenocytes culture supernatant levels cytokines IL-4 (Boster, Wuhan, China), IL-5 and IFN-γ (Sino Biological Inc, Beijing, China) were determined by ELISA with commercial reagent kits following the manufacturer’s instruction.

Cytokine production was determined with Enzyme-Linked Immunosorbent Assays (ELISAs) using commercial kits according to the manufacturers’ instructions. Interleukin-1beta (IL-1β) (cat no: EK0393) and IL-4 (cat no: EK0406), levels were measured using anti-rat ELISA kits from BosterBio (Pleasanton, CA, USA). IL-6 (cat no: KHC0061) and Tumor Necrosis Factor-alpha (TNF-α) (cat no: KRC3011) were obtained from Invitrogen (Carlsbad, CA, USA). Microtiter plates were read at 450 nm using the CA-2000 ELISA microplate reader (CIOM Medical Co., Ltd., Changchun, China). Using linear regression analysis, cytokine levels were calculated from standard curves of recombinant cytokines.