Mouse ige elisa set

At sacrifice, about 1 mL of venous blood was collected from vena cava under anesthesia with 2 to 3% isoflurane (Hana Pharm. Co., Hwaseong, Republic of Korea) in the mixture of 70% N₂O and 28.5% O₂, and serum was separated by centrifuging at 15,000 rpm for 10 min under 4°C, using clotting activated serum tube. Total IgE levels in serum were determined by sandwich ELISA using the mouse IgE ELISA set (BD Biosciences, San Diego, CA, USA) according to previous methods [4 (link), 6 (link)]. Briefly, plates were coated with capture antibody in ELISA coating buffer and incubated overnight at 4°C. Plates were washed with PBS-Tween 20 (0.05%) and subsequently blocked (10% FBS in PBS) for 1 hr at 20°C. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates and plates were incubated for 2 hrs at 20°C. After washing, biotin-conjugated anti-mouse IgE and streptavidin-horseradish peroxidase conjugate were added to the plates and plates were incubated for 1 hr at 20°C. Finally, tetramethylbenzidine substrate solution was added to the plates and after 15 min of incubation in the dark, a 2NH₂SO₄ solution was added to stop the reaction. Optical densities were measured at 450 nm on an automated ELISA reader (Tecan; Männedorf, Switzerland).

Upon euthanasia, blood was harvested through a cardiac puncture and serum was collected. For IgGs, ELISA plates were coated with 50 μg of MSS, blocked with 10% FBS in PBS and incubated with serum dilutions. Samples were then incubated with either anti‐mouse IgG₁‐HRP or IgG_2a‐HRP (BD Biosciences, San Diego, CA). Total IgE were measured using a Mouse IgE ELISA Set (BD OptEIA) according to the manufacturer's instructions. The reactions were revealed with BD OptEIA TMB (BD Biosciences) and stopped with 1N HCl.

DMEM/F-12 Medium, knockout serum replacement, glutamine, β-mercaptoethanol, nonessential amino acids, and human recombinant bFGF were purchased from Invitrogen Corporation (Carlsbad, CA, USA). EBM^TM-2 Basal Medium and EGM^TM-2 MV Microvascular Endothelial Cell Growth (EGM2-MV) Medium SingleQuots^TM supplements were purchased from Lonza (Basel, Switzerland). Trypsin-EDTA (0.25%) was purchased from Gibco (Waltham, MA, USA). Sodium chloride, 1-chloro-2,4-dinitrobenzene (DNCB), eosin Y, toluidine blue O, hematoxylin, and hydrochloric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Total RNA Extraction Kit was purchased from iNtRON (Gyeonggi, Korea). High Capacity cDNA Reverse Transcription Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FastStart Essential DNA Green Master and serum-containing medium were purchased from Roche (Basel, Switzerland). Ammonia was purchased from JUNSEI (Chuo-ku, Tokyo). Anti-F4/80 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse IgE ELISA Set was purchased from BD (Franklin Lakes, NJ, USA).