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20 protocols using ELISA

eNOS enzymatic activity was determined by measuring the conversion of l-[3H]-arginine (4 µCi/ml) to l-[3H]-citrulline, separated in formic acid digests on Dowex 50W8 columns. eNOS activity was expressed as l-NAME inhibitable l-[3H]-citrulline production. cGMP production was assessed using a chemiluminescent ELISA (Arbor Assays, Ann Arbor, MI, USA) in the presence of the phosphodiesterase inhibitor IBMX (0.5 mM), with or without the eNOS inhibitor l-NAME (100 µM).
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Basal plasma CORT was quantified via a colorimetric enzyme-linked immunosorbent assay (ELISA) (Arbor Assays, Ann Arbor, MI) according to manufacturer protocol.
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The ability of strains to effectively deliver heat-labile toxin to host cells, cultures were grown overnight then used to infect target Caco-2 intestinal epithelial cell monolayers. After incubation for 2.5 hours at 37 °C, 5% CO2 supernatant was removed and replaced with pre-warmed tissue culture media, and incubated for another 2 hours. Monolayers were then lysed and cAMP was determined using by ELISA (Arbor Assays).
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Total cell lysates were prepared from brain tissue of lean, HFD and GaELNs treated HFD mice and BV2 cells were treated with metabolites derived from brain tissues. The level of cGAMP in cell lysates was measured by ELISA according to manufacturers' instructions (Arbor Assays, An Arbor, MI). The results were normalized based on total protein concentration.
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Bacteria were inoculated from −80 °C frozen stock, and grown overnight in casamino acids-yeast extract medium (CAYE) medium at 37 °C with shaking. Overnight cultures were centrifuged at 16,000 rpm for 10 minutes, and the clarified supernatants were used for ELISA (Arbor Assays) as previously described69 (link). In brief, 100 µl of culture supernatant was applied to the ELISA plate coated with 0.1 ug of GM1 ganglioside overnight at 4 °C. Plate was incubated at room temperature for 2 hours, then washed with PBS-T (PBS with 0.05% tween 20), followed by blocking with 200 ul of 1% BSA for 1 hour. After blocking, 100 µl of affinity purified anti-LT-B subunit antibody was added in 1:1000 dilution, and incubated for 1 hour at 37 °C. Plates were then washed with PBS-T, followed by the addition of 100 µl of secondary antibody (anti-rabbit-IgG) at a 1:5000 dilution, incubated at 37 °C for 0.5 hours, then washed again with PBS-T. Then 100 µl of TMB substrate mixture was added to each well and the optical density at 650 nm was determined immediately and every 40 seconds thereafter for kinetic analysis. ETEC strains H10407 and jf565 were used as positive and negative controls, respectively.
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Oxytocin immunoreactivity in plasma, ascites, and cell culture supernatant was analyzed by ELISA (Arbor Assays, Ann Arbor, MI) following extraction of samples using C18 Sep-Pak columns (Waters; Milford, MA) as described (Szeto et al., 2011 (link)). Oxytocin immunoassay was performed following manufacturer’s instructions; the lower limit of detection was approximately 1 pg/well and the intra and inter-assay coefficient of variance (CV) were < 8% and < 12%, respectively.
Interleukin-6 in plasma and ascites was analyzed by ELISA (R&D Systems, Minneapolis, MN) and in cell culture supernatants using an ELISA from BD Biosciences (Cat # 555220; Franklin Lakes, NJ). The minimum detectable level was less than 7 pg/mL and inter-assay CV ranged from 3.3% to 6.4%. Samples with interleukin-6 levels below the sensitivity of the regular assay were quantified using the R&D high sensitivity ELISA; intra-assay CV was less than 10%.
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MC38-Ovadim was implanted in wild type C57BL/6 mice and either Metyrapone (50mg/kg; Fisher Scientific), Corticosterone (2.5mg/kg ) or vehicle control PBS (Gibco) was administered intra-tumorally on Day 5,6,7 and 9 post-tumor implantation. In some experiments, MC38-Ovadim tumor explants or sorted linCD45+CD24 cells were cultured in the presence or absence of Metyrapone (25 or 50 ng/ml) for 24hrs. Supernatants were harvested and Corticosterone measured by ELISA (Arbor Assays).
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To clarify the mechanisms of YKSCH, mice were randomly divided into 3 groups: control group (n = 5), social isolation stress group (n = 5), social isolation stress + YKSCH (1,500 mg/kg)-treated group (n = 5). Mice were subjected to social isolation stress for 4 weeks with or without YKSCH treatment. One hour after the last treatment with YKSCH, mice were decapitated, and brains quickly removed from the skull, briefly washed in ice-cold saline, laid on a cooled (4°C) metal plate, and both the OB and PFC were rapidly dissected out (Figure 1D). The tissues (10 mg) were added to 100 μL ethanol and shaken vigorously for 30 min, and then centrifuged at 5,000 rpm for 15 min. Supernatants were removed and transferred to new clean tubes and evaporated to dryness under nitrogen. Then, 100 μL ethanol and 400 μL assay buffer were added to dissolve the sample and vortexed well. The extracted samples were used for immunoassays. The ALLO content in the OB and PFC was assayed using ELISA (Arbor Assays, MI, USA).
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Tail blood samples were collected via heparinized capillary tubes. All mice were previously habituated to handling for at least 7 consecutive days. Plasma corticosterone was measured using an ELISA (Arbor Assays) according to the manufacturer’s instructions. For ACTH measurements, mice were decapitated and trunk bloods were collected in lavender EGTA-coated tubes. All samples were kept on ice and centrifuged at 4 °C within minutes of collection. ACTH was measured using an ELISA (MD Bioproducts) according to the manufacturer’s instructions.
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For urinary corticosterone measurements, all urine was collected from mice over a 24 h period. Corticosterone was then measured by ELISA (Arbor Assays: K014-HI). To control for differences in overall urine concentration, urinary creatinine was measured by ELISA (Enzo Life Sciences: ADI-907-030A). A ratio of corticosterone:creatinine was then generated.
For plasma corticosterone measurements, tail blood or trunk blood following mouse decapitation was collected and immediately placed at 4 °C. Blood was centrifuged at 20,000 rcf at 4 °C and plasma was frozen at −20 °C. Plasma corticosterone was determined by I-125 radioimmunoassay (MP Biomedical, Solon, OH, USA) [26] (link). The intra-assay and inter-assay CVs for plasma corticosterone were 7.6% and 13.3%, respectively. Serum cardiac troponin I (cTnI) was measured by ELISA (Life Diagnostics: CTNI-1-HS).
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