To quantify human IL-6 and IFN-β production, specific capture ELISAs were performed. A rat anti-human IL-6 capture antibody (BD Pharmingen, cat# 554543; Clone Mq2-13A5, 2 μg/ml) and a biotinylated rat anti-human IL-6 detection antibody (BD Pharmingen, cat# 554546; Clone MQ2-39C3, 2 μg/ml) were used in IL-6 ELISAs. While, a polyclonal rabbit anti-human IFN-β capture antibody (Abcam, cat# ab186669, 0.25 μg/ml) and a biotinylated polyclonal rabbit anti-human IFN-β detection antibody (Abcam, cat# ab84258, 0.25 μg/ml) were used in IFN-β ELISAs. Bound antibody was detected using streptavidin-HRP (BD Biosciences) followed by the addition of tetramethylbenzidine substrate. H2SO4 was used to stop the reaction and absorbance was measured at 450 nm. A standard curve was generated using dilution of recombinant cytokines for IL-6 (BD Pharmingen) and IFN-β (Abcam). The cytokine concentration in cell supernatants was determined by extrapolation of absorbance to the standard curve.
Ifn β
Manufactured by Abcam
Sourced in United States
IFN-β is a recombinant human interferon beta protein. Interferons are a group of signaling proteins involved in the innate immune response against viral infections and other biological activities.
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Lab products found in correlation
Ifn β, by OriGene (2 mentions)
Ifn β, by PBL Assay Science (2 mentions)
Neuroporter, by Genlantis (2 mentions)
Pli 100, by Harvard Apparatus (2 mentions)
Clone mq2 13a5, by BD (1 mentions)
Recombinant cytokines for il 6, by BD (1 mentions)
Streptavidin hrp, by BD (1 mentions)
Tgf β, by Abcam (1 mentions)
Sybr green assay, by Roche (1 mentions)
Pai 1 total mouse elisa kit, by Abcam (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Mmp 9, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Ripa lysis buffer, by Keygen Biotech (1 mentions)
Ifn β, by BioLegend (1 mentions)
Anti ifn β antibody, by Abcam (1 mentions)
Female c57bl 6 mice, by Charles River Laboratories (1 mentions)
Anti mouse f4 80 apc, by Thermo Fisher Scientific (1 mentions)
13 protocols using ifn β
1
Quantification of Human IL-6 and IFN-β
2
SERPINE1 Expression and Secretion Kinetics
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To determine SERPINE1 mRNA expression and protein secretion kinetics, we treated A549 cells or HAE cultures with the following conditions: IFN-β (Abcam) at 1pmol/ml, TGF-β (Abcam) at 1 pg/ml, IAV WSN/33 infection at MOI 1 (A549) or 0.1 (HAE). mRNA levels, normalized to housekeeping gene RPS-11, were determined by qRT-PCR (SuperScript III First Strand Synthesis System, Life Technologies) and SYBR green assay (Roche) as described previously. Primer sequences can be found in Table S3 . Total PAI-1 protein levels from cell supernatants or cell lysates (1% Triton X-100 in PBS, sonication for 10 min) were measured by Human PAI-1 Platinum ELISA (BD Biosciences). mPAI-1 levels from mouse lung homogenates were measured by PAI-1 total mouse ELISA kit (Abcam).
3
Western Blot Analysis of Protein Expression
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Cells or tissues were lysed in RIPA Lysis Buffer supplemented with a cocktail of protease inhibitors (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), followed by centrifugation at 14,000 × g for 10 min. After centrifugation, the cell lysates were collected and the protein concentrations measured. Proteins (20–30 µg) were resolved by SDS-PAGE, and then transferred to PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% BSA at room temperature for 3 h, and then incubated with primary antibodies in 3% BSA/TBST at 4°C overnight, followed by incubation with secondary antibodies (Auragene Bioscience Corp., Inc., Changsha, China) at room temperature for 1 h. The protein signals were detected by the ECL method. The following antibodies were used for western blotting: NAC1, GAPDH, β-actin, IFNβ, RANK, MMP9 and PKM2 antibodies, all purchased from Abcam.
4
Murine Model of Bacterial Pneumonia
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Female C57BL/6 mice (aged 8 to 14 wk, Charles River Laboratories) were housed in pathogen-free conditions. The Animal Care Committee of the Maisonneuve-Rosemont Hospital approved the protocols (permit Nos. 2015-31 and 2019-1765). Mice were injected intratracheally with 5 × 106 live E. coli with or without simultaneous injection of CpG DNA (1 μg/g, intraperitoneally [i.p.]) (9 (link)). At 6 h postinfection, mice were treated with IFN-β (BioLegend) (2.5 ng/g, i.p.), 15-epi-LXA4 (125 ng/g, i.p.), or 17-epi-RvD1 (25 ng/g, i.p.), as informed from previous studies (9 (link), 51 (link), 52 (link)). Some mice received cyclosporin H (5 μg/g, i.p.) or WRW4 (1 μg/g, i.p.) (28 (link)) 10 min before IFN-β or were injected with an anti–IFN-β antibody (1 μg/20 g, i.p., Abcam) or IgG1 before intratracheal challenge with E. coli.
5
Investigating Macrophage Signaling Pathways
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IFN-β were purchased from Abcam (Cambridge, MA, USA) and anti-mouse IFN-β was purchased from BioLegend (San Diego, CA, USA). Anti-mouse SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Escherichia coli LPS from serotype 0111:B4, mouse macrophage colony-stimulating factor (MCSF), thioglycollate, anti-β-actin antibody, and anti-α-tubulin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse CD11b-FITC and anti-mouse F4/80-APC were purchased from eBioscience (San Diego, CA, USA). LY294002, wortmannin), SB203580, PD098059, SP600125, PDTC, JAK1 inhibitor, and JAK3 inhibitor were purchased from Calbiochem (San Diego, CA, USA).
6
Liver Protein Extraction and Western Blot
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The liver stored at −80 °C was weighed and added with RIPA lysis buffer (Biyuntian, China), the supernatant absorbed by centrifugation was the total protein of the liver after sufficient vortex lysis on ice. The samples’ protein concentration was test by BCA protein assay kit (Biyuntian, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20 μg) were used to separate the samples and polyvinylidene fluoride (PVDF) membrane (Merck Millipore, USA) was used to transferred the proteins. The transferred membrane is incubated and bound with a primary antibody (TLR3 (rabbit polyclonal antibody, Abcam, USA) TRIF (rabbit polyclonal antibody, Abcam, USA) TBK1(rabbit polyclonal antibody, Abcam, USA) IRF3(rabbit monoclonal antibody, Abcam, USA) IFNβ (rabbit polyclonal antibody, Abcam, USA)), then the membrane is further incubated and bound with a second antibody conjugated with the appropriate horseradish peroxidase. Finally, protein bands were displayed using ECL chromogenic solution (Biyuntian, China).
7
Quantifying Cytokine Production in Cell Lines
8
Quantification of IFN-β in Poly(I:C)-Treated PBMCs
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The isolated PBMCs were cultured in 6-well microtiter plates (1 106 cells/well in 2 mL RPMI 1640 media; Sigma, Poole, United Kingdom) at 37 °C with 5 % CO2. The cells were treated with 25 μg/ml of poly(I:C) (Sigma-Aldrich, MO, USA; catalog no. P1530) for a duration of 24 h. The level of IFN-β protein production was determined in the culture supernatant using a commercially available human ELISA kit (IFN-β, Abcam, Cambridge, MA, USA; catalog no. ab278127). Assays were performed strictly following the manufacturer's instructions. Each sample was assayed in duplicate, and values were expressed as the mean of 2 measures per sample. One-way analysis of variance (ANOVA) and post hoc Tukey multiple comparison analyses were applied.
9
Subretinal Delivery of Genetic Agents in Mice
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Subretinal injections (1 μl) in mice were performed using a Pico-Injector (PLI-100, Harvard Apparatus) or using a 35-gauge needle (Ito Co. Fuji, Japan). In vivo transfection of plasmids expressing Alu sequences (pAlu)67 (link),68 (link), empty control vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)57 ,69 (link), wildtype mouse gasdermin D (pGSDMD-WT), the p30 cleavage incompetent mutant mouse gasdermin D ((pGSMDD-D276A)19 (link), IFN-β (Origene Cat# MR226101), or mtDNA (10 ng) was achieved using 10% Neuroporter (Genlantis) as previously described4 (link),5 (link). In vitro transcribed Alu RNA (0.15–0.3 μg/μl), IFN-β neutralizing antibody (10 ng; Abcam Cat# ab24324), control isotype IgG, recombinant IL-18 (100 ng/μl, MBL Cat#B002-5), or IFN-β (500 mUnit/μl, PBL Cat#12410-1) were administered via subretinal injection4 (link),5 (link). Similarly, to knock down Dicer1, 1 μl of cholesterol conjugated siRNA (1 μg/μl) targeting mouse Dicer1 or scrambled control siRNAs were injected (Dicer1 siRNA sense- CUCUGUGAGAGUUGUCCdTdT; Control siRNA sense- UAAGGCUAUGAAGAGAUdTdT). The eye used for active versus control injection was chosen randomly.
10
Subretinal Delivery of Genetic Agents in Mice
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