Ifn γ elisa kit

DC260126 (MCE, New Jersey, USA) was employed to antagonize GPR40 expression. Y-27632, the inhibitor of ROCK, was obtained from Tocris Bioscience (Bristol, UK). High-fat diet (NO. MD12032) was purchased from Medicience Ltd (Jiangsu, China). Methacholine (Mch), Ovalbumin (OVA) and DMSO were bought from Sigma-Aldrich (St. Louis, MO), TRIzol reagents were used to extract total RNA (Takara, Otsu, Shiga, Japan). Oleic acid (OA) was purchased from Sigma-Aldrich Company, USA. The following antibodies: RhoA (1:5000, #AB187027, rabbit polyclonal, Abcam), ROCK1 (1:2000, #ab45171, rabbit polyclonal, Abcam), GPR40 (1:1000, #DF2745, rabbit polyclonal, Affinity Biosciences), GAPDH (1:5000, #AF7021, rabbit polyclonal, Affinity Biosciences) were applied to the western blot analysis. Mouse FFAs, IL-4, IL-5, IL-13, IL-1β, TNF-α, and IFN-γ ELISA kits were obtained from eBioscience (San Diego, CA).

Peripheral blood mononuclear cells were isolated from heparinized blood by density gradient purification over Lymphoprep and stimulated with 5 ng/Ml Phorbol 12-myristate 13-acetate (PMA) (P-8139, Sigma, Welwyn Garden City, UK ) and 0.1 ug/mL ionomycin (I-0634, Sigma, Welwyn Garden City, UK) for 3.5 h or left unstimulated. Supernatants were assayed for IL-17 or IFN-γ levels by IL-17 ELISA kit (KAC1591, Invitrogen, Paisley, UK) and IFN-γ ELISA kit (KHC4021, Invitrogen, Paisley, UK) according to the manufacturer’s instructions. As the unstimulated samples showed undetectable levels of protein, readings included in the analysis were from PMA/ionomycin stimulated samples.

Blood was extracted from mice after they were killed, and serum was obtained by centrifuging the blood collected (Cence, Changsha, Hunan, China). Histamine and IgE were determined by using histamine and IgE Enzyme-linked immunosorbent assay (ELISA) kit (Jiancheng Bioengineering Institute, Nanjing, Jangsu, China) according to the instructions.
Nasal lavage fluid samples were collected from mice by washing their noses at the end of animal experiment. IL-2, IL-4, IL-17 and IFN-γ from nasal lavage fluid were detected by IL-2, IL-4, IL-17 and IFN-γ ELISA kit (Invitrogen, Carlsbad, California, U.S.A.), respectively.

The production of IL-12p40 and IFN-γ were measured using a murine IL-12/IL-23p40 ELISA Kit (Invitrogen, Carlsbad, CA) and a IFN-γ ELISA Kit (Invitrogen) according to the manufacturer’s instruction. Briefly, 96 well ELISA plates (Corning Costar, Corning, NY) were coated with anti-cytokine capture antibody in coating buffer (Invitrogen) and incubated overnight at 4°C. The plates were washed 3 times in PBS containing 0.05% Tween-20 (PBST) then blocked with diluent (Invitrogen) for 1 hr at room temperature. After washing the plates once in PBST, sample supernatants as well as recombinant standard was added and incubated overnight at 4°C. The plates were washed 5 times in PBST then anti-cytokine biotin detection antibody was added and plates were incubated at room temperature for 1 hr. The plates were washed 5 times in PBST, avidin-horseradish peroxidase was added and plates were incubated for 30 min at room temperature. The plates were washed 5 times in PBST and 3,3’,5,5’-Tetramethylbenzidine (TMB) was added and plates incubated for 20 min. The reaction was quenched with 2 M H₂SO₄ and plates were read at 450 nm on an iMark ELISA reader (Bio-Rad, Hercules, CA).

The FINM (2 × 10⁵ NKs + 2 × 10⁴ WJ-MSCs/well) was seeded together with different MM cell lines (2 × 10⁴/well) in a 96-well plate in full DMEM medium (150 μL/well). 100 μL of conditioned medium was collected from each well after 7 days of co-culture. IFN-γ secreted by NKs was quantified by IFN-γ ELISA Kit (Invitrogen, Waltham, MA, USA) following manufacturer’s protocol. Absorbance (450 nm) was measured using the micro-plate reader Infinite^® F Plex (Tecan, Männedorf, Switzerland, version 3.9.0.1). IFN-γ concentrations were calculated based on kit’s standard curve measured alongside the samples.
In a 96-well plate, BZ-treated and untreated MM cells were seeded (4 × 10⁴ cells/well) in 100 μL of complete DMEM medium. Both conditions were treated with recombinant human IFN-γ (Peprotech, London, UK; 100 IU/mL) and left incubated for 3 days. BLI peak values were taken from each well (Infinite^® F Plex, Tecan, Männedorf, Switzerland, version 3.9.0.1), and cell viability (%) was calculated using following equation.

The indicated brain regions of male mice were disrupted with a homogenizer and analyzed for IFN-γ, ACh and DA content using a sandwich ELISA, following the manufacturer’s instructions (IFN-γ ELISA kit (KAC1231) was from Life technologies Invitrogen (Carlsbad, CA, USA); ACh ELISA kit (#E4453-100) was from BioVision (Milpitas, CA, USA); DA ELISA kit (KA1887) was from Abnova (Walnut, CA, USA). Briefly, 96-well ELISA microplates were coated with specific monoclonal Ab. Samples or standard were added at the appropriate dilution and incubated for 2 h at room temperature. After careful washing, biotinylated goat anti- IFN-γ, ACh or DA were added to each well; horseradish-peroxidase was used as secondary Ab and optical density was read at 450 nm.
In order to quantify ACh released by NK cell in vitro, purified murine and human NK cells were activated O/N in IL-15 (20 ng/ml) and then co-incubated for 48 h with PMA/ionomycin (1:100), IL-15 (50 ng/ml), IL-2 (50 ng/ml), or with primary microglia at ratio 1:1. Then medium was collected and analyzed for ACh ELISA kit (#E4453-100).