CaCo-2 and HT-29 cells were plated in 12-well plates at 1 × 106 cells/well or 1.2 × 106 cells/well, respectively. Eighteen-hours post plating, IECs were mock-treated with IFN-buffer (0.1% BSA in phosphate buffer saline, PBS) or with IFNλ1 or IFNλ2 dissolved in PBS containing 0.1% BSA (both at a concentration of 100 ng/mL; Preprotech, Rocky Hill, NJ, USA) for 6 h or 24 h. IFN-α2b, also diluted in PBS-0.1% BSA, was used at a concentration of 1000 U/mL (Intron A; Merck Sharp & Dohme, Stockholm, Sweden). The concentrations of the IFNλs (100 ng/mL) and IFNα (1000 U/mL) used were based on previous studies performed in our group ([23 (link),25 (link)] and data not shown) and from other studies that demonstrate cellular responses with no accompanying adverse effects [35 (link)]. Six or 24 h post treatment, either mRNA was isolated and used to determine changes in gene expression via real-time reverse transcription-polymerase chain reaction (RT-PCR) or protein was extracted and alterations in protein expression were studied by Western blotting. In virus infection experiments, cells were pre-treated with IFNs for 24 h, infected with CVB3 (see Section 2.4 ), and then cultured for 24 h with IFNs. The supernatant and cells were then collected and viral titers measured by plaque assay.
Intron a
Manufactured by Merck Group
Sourced in United States
Intron A is a laboratory product manufactured by Merck Group. It is a recombinant form of human interferon alpha-2b, which is a naturally occurring protein involved in the body's immune response. Intron A is used as a research tool in molecular biology and biochemistry experiments, where it can be utilized to study the role of interferon alpha-2b in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Begm with supplements, by Lonza (2 mentions)
Ifn λ3, by R&D Systems (2 mentions)
Ifn λ1, by Thermo Fisher Scientific (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Itaq sybr green, by Bio-Rad (1 mentions)
Iscript, by Bio-Rad (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Lps eb, by InvivoGen (1 mentions)
Truculture tubes, by Merck Group (1 mentions)
Trizol ls, by Merck Group (1 mentions)
Pegasys, by Roche (1 mentions)
Pfu turbo dna polymerase, by Agilent Technologies (1 mentions)
Dna ligation kit, by Takara Bio (1 mentions)
Bamh1, by Toyobo (1 mentions)
Plasmid kits, by Qiagen (1 mentions)
Il 12, by Miltenyi Biotec (1 mentions)
Anti cd56 apc, by Beckman Coulter (1 mentions)
Anti ifn γ fitc, by BD (1 mentions)
Anti tcr vα7.2 pe 3c10, by BioLegend (1 mentions)
14 protocols using intron a
1
Interferon Signaling in Intestinal Epithelial Cells
2
IFN Treatment of Human Epithelial Cells
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All IFNs used are listed in Table S5 . The IFN-treated NHBE cells were previously described22 (link). Briefly, the cells were cultured in BEGM with supplements (Lonza), plated in 6-well plates and utilized at ~70% confluence (typically after 10–11 days with media change every 2 days). Cells were left untreated or treated with IFN-α2b (INTRON A, Merck, 100 IU/ml) or IFN-λ3 (R&D Systems, 100ng/ml) for 24 hrs. Cells were washed with PBS, resuspended in TRIzol (Thermo Fisher) and stored at −80°C for future RNA isolation. Total RNA was extracted with Direct-zol mini RNA isolation kits (Zymo Research). The IFN treatment of human colon and ileum organoids was previously described23 . Briefly, cells were seeded 24 hrs prior to IFN treatment to reach a confluency of 70% at the time of treatment. Media was removed from cells and replaced with a cocktail of IFN-λ1–3 (100ng/mL of each for a total of 300ng/mL). Media was removed 24 hrs post-treatment, and RNA was extracted using the RNAeasy kit (Qiagen). 250ng of RNA was used to prepare cDNA using iSCRIPT (BioRad) and qRT-PCR was performed using iTaq SYBR Green (BioRad). T84 and Caco-2 cells were treated with IFN-γ (2 ng/ml) for 48 hrs, followed by cell harvesting, RNA extraction and expression analysis.
3
Combination Therapy for Japanese Encephalitis
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The use of combination treatment of intravenous immunoglobulin (IVIG), oral ribavirin, and subcutaneous interferon-α2b was initiated as soon as the KDCA report suggested laboratory confirmation of the diagnosis of JE and if the neurological symptoms and signs including altered mental state, confusion, and brainstem signs such as diplopia could not be explained by an etiology other than JEV infection. Another infectious etiology (another virus, bacterium, or fungus) and vascular, inflammatory, and tumorous etiologies also needed to be ruled out to diagnose with JE.
Patients with JE received intravenous human immunoglobulin G for 5 consecutive days at a dosage of 400 mg/kg/day. Ribavirin (Viramid, Ilsung Pharmaceuticals, Seoul, Korea) was administered orally at a dosage of 400 mg every 12 h for 14 consecutive days. Interferon-α2b (Intron A, Merck & Co, Kenilworth, NJ, USA) was given by subcutaneous injection at a dosage of 3 million units per day for 2 weeks. Some of the patients with JE did not receive all components of the combination therapy due to medical contraindication, economic issues, or personal rejection. All patients received appropriate supportive management as required, including intravenous hydration, nutritional support, oxygenation, mechanical ventilation, or antibiotics, and intensive care.
Patients with JE received intravenous human immunoglobulin G for 5 consecutive days at a dosage of 400 mg/kg/day. Ribavirin (Viramid, Ilsung Pharmaceuticals, Seoul, Korea) was administered orally at a dosage of 400 mg every 12 h for 14 consecutive days. Interferon-α2b (Intron A, Merck & Co, Kenilworth, NJ, USA) was given by subcutaneous injection at a dosage of 3 million units per day for 2 weeks. Some of the patients with JE did not receive all components of the combination therapy due to medical contraindication, economic issues, or personal rejection. All patients received appropriate supportive management as required, including intravenous hydration, nutritional support, oxygenation, mechanical ventilation, or antibiotics, and intensive care.
4
Whole Blood Stimulation and Cytokine Analysis
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For whole blood stimulation, TruCulture tubes (RBM) containing Poly:IC (20 μg/mL), R848 (1 μM), LPS-EB (ultrapure) (10 ng/mL) (all Invivogen), and IFN-α2 (Intron A, Merck) dissolved in 2 mL of buffered media were batch produced as previously described38 (link). Tubes were thawed at room temperature and 1 mL of fresh blood was distributed into each tube within 15 min of collection. Tubes were mixed by inverting them and incubated at 37 °C for 22 h in a dry block incubator. After the incubation time, a valve was manually inserted into the tube to separate the supernatant from the cells. The supernatant was collected, aliquoted, and immediately stored at −80 °C for protein secretion analysis. Cell pellets of the TruCulture tubes were resuspended in 2 mL of Trizol LS (Sigma) and tubes were vortexed for 2 min at 2000 rpm and stored at −80 °C for gene expression analysis.
5
HCV Infection in Chimeric Mouse Model
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We used chimeric mice with human albumin concentrations equal to or greater than 5,000μg/mL at 8 weeks post transplantation for the HCV infection studies. Chimeric mice received a single intravenous injection of human patient HCV-positive serum (approximately 105 genome equivalence). The collection and use of human sera in this study were approved by the Health Research Ethics Board, University of Alberta (Study ID: Pro00001040, Study Title: Blood Samples as a source of Hepatitis B or Hepatitis C viruses for studies in an animal model of HBV or HCV). Each patient has given written informed consent to donate blood to be used in this study.
We administered exogenous human IFNα subcutaneously (s.c.) for 14 days at 1,350 IU/g body weight/day using human IFNα-2b (INTRON® A, MERCK). Six hours after the last IFNα injection, we euthanized the mice and collected samples/tissues. We gave the same volume of saline to chimeric mice in the control group. For groups treated with pegylated human IFNα-2b (Pegasys, Roche), we administered 875ng once a week by s.c. injection. After 4 weeks, we discontinued treatment and monitored the animals for relapse for 2 weeks before termination.
We administered exogenous human IFNα subcutaneously (s.c.) for 14 days at 1,350 IU/g body weight/day using human IFNα-2b (INTRON® A, MERCK). Six hours after the last IFNα injection, we euthanized the mice and collected samples/tissues. We gave the same volume of saline to chimeric mice in the control group. For groups treated with pegylated human IFNα-2b (Pegasys, Roche), we administered 875ng once a week by s.c. injection. After 4 weeks, we discontinued treatment and monitored the animals for relapse for 2 weeks before termination.
6
Plasmid Construction and Enzyme Usage
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PfuTurbo DNA Polymerase was obtained from Agilent Technologies (Santa Clara, CA). The restriction enzymes of BamH1 and EcoR1 were purchased from Toyobo Co., Ltd. (Osaka, Japan). The restriction enzymes of Ava1, Sal1 and Xho1 and DNA Ligation Kit were purchased from Takara BIO Inc. (Kyoto, Japan). QIAGEN Plasmid Kits were purchased from QIAGEN, Inc. (Hilden, Germany). INTRON® A was obtained from Merck & Co., Inc. (Kenilworth, NJ, USA). Mannan was purchased from Nacalai Inc. (Kyoto, Japan). All other chemicals and reagents used were of the highest commercially available quallity, and all solutions were made using deionized and distilled water.
7
Intraperitoneal Vaccine and Interferon Therapy for Tumor
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Upon arrival at the veterinary clinic, DC vaccine aliquot one of four, suspended in 2 ml normal saline, was injected intraperitoneally under ultrasound guidance into the abdominal cavity in the vicinity of the original tumor. Concurrent with vaccine administration, five million units human interferon alpha (Intron A, Merck & Company, Inc, Kenilworth, NJ) was administered subcutaneously. Subsequently, five million units of veterinary type I interferon omega (Vibragen Omega, VioVet, Ltd, Bedfordshire, United Kingdom) was administered every other day for a total of 19 additional administrations. Echo/ultrasound of the heart was performed as were chest radiographs, complete blood count (CBC), and clinical chemistry.
8
Quantifying IFN-γ Production in Immune Cells
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The percentage of cells producing IFN-γ was measured by flowcytometry using various stimuli. For each condition, 500,000 cells were stimulated in a 24 wells plate with IL-12 (0.25 ng/ml, Miltenyi), IL-18 (50 ng/ml, MBL) and IFN-α (2500 IU/ml, Intron-A, Merck). For all conditions, cells were stimulated for 16 hours at 37°C at 5% CO2. Brefeldin A (10 μg/ml, Sigma) was added and the cells were incubated for another 3 hours. Cells were stained with anti-CD3-PerCp-Cy5.5 (UCHT1), anti-CD4-APC-H7 (SK3, both BD biosciences), anti-CD69-PE-Cy7 (TPI.55.3), anti-CD161-PB (HP-3G10, both eBiosciences), anti-TCR Vα7.2-PE (3C10, Biolegend), anti-CD56-APC (N901, Beckman) and Live/ dead marker (Aqua, Life technologies). Cells were fixed, permeabilized and stained with anti-IFN-γ-FITC (25723.11, BD Biosciences). Cytokine-producing cells were detected by flow cytometry (Canto-II, BD). Quadrants were set on low or absent expression on lineage negative cells. Data was analyzed using FlowJo version 10.1 (Tree Star Inc).
9
Cytokine-induced STAT Phosphorylation
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For the analysis of STAT phosphorylation, cells were stimulated with the indicated doses of recombinant IFNα-2b (Intron-A, Merck), IFNγ, IL-2, IL-4, and IL-6, IL-13 (Biolegend) for 15 minutes and then lysed for western blotting. For the analysis of gene induction, cells were stimulated for 8 hours, after which cells were lysed for RNA isolation. For JAK inhibitor treatment, cells were incubated with ruxolitinib (Selleckchem S1378), filgotinib (SelleckchemS7605) or tofacitinib (Selleckchem S5001) at the indicated doses for 4 hours.
10
Isolation and Activation of Human T Cells
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PBMCs were isolated by density centrifugation through Ficoll-Hypaque PLUS (GE Healthcare Life Sciences). Pan T cells were isolated by negative selection using Pan T Cell Isolation kit (Miltenyi Biotec) to ≥98% purity. Pan T cells were stimulated with anti-CD2/CD3/CD28 coated beads from the T Cell Activation/Expansion kit (Miltenyi Biotec), at a bead to cell ratio of 1:1. T cells were cultured in RPMI medium supplemented with 10% FBS (Gibco), 100 U/ml recombinant human IL-2 (Aldesleukin, Prometheus), 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Gibco), and 55 µM 2-ME (Sigma-Aldrich). Where indicated, previously activated cycling T cells were treated with 100 IU/ml recombinant human IFN-alpha-2b (Intron A; Merck) for 20 h to induce MDA5 expression.
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