FT3-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) and supplemented with 10% fetal calf serum and 2 mM GlutaMAX (Life Technologies, Carlsbad, CA). Cells were cultured in a humidified incubator at 37°C and 5% CO2. Three technical replicates of 1 × 107 FT3-7 cells were harvested in lysis buffer [150 mM KCl, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1% Triton X-100, 5 mM DTT, Complete protease inhibitor mixture (Roche), and 100 U/mL RNaseOUT (Life Technologies)]. Lysates were centrifuged for 30 min at 17,000 × g at 4°C and filtered through a 0.22-um filter. Filtrates were incubated with anti-human AGO2 mAb (RN003M, MBL International, Woborn, MA) or isotype control IgG (Abcam, Cambridge, England) at 4°C for 2 h, followed by addition of 30 μL of Protein G Sepharose (GE Healthcare) for 1 h. The Sepaharose beads were washed three times in lysis buffer and RNA extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany).
Igg isotype control
Manufactured by Abcam
Sourced in United Kingdom, United States
The IgG isotype control is a laboratory reagent used to establish background staining levels in immunoassays. It serves as a reference for non-specific binding of antibodies, allowing for accurate interpretation of target-specific signal.
Automatically generated - may contain errors
Lab products found in correlation
Lowcell chip kit, by Diagenode (2 mentions)
Luciferase assay kit, by Promega (2 mentions)
Ab105385, by Abcam (2 mentions)
Glomax luminometer, by Promega (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Anti cd73 pe, by BD (2 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Rn003m, by MBL Life Science (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Cxcl13, by R&D Systems (1 mentions)
Facsdiva 6, by BD (1 mentions)
Monoclonal anti ffa1 antibody rabbit, by Abcam (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Anti cd29, by BD (1 mentions)
Anti cd63, by BD (1 mentions)
Anti cd90 pe cy7, by BD (1 mentions)
Anti cd44, by Bio-Rad (1 mentions)
17 protocols using igg isotype control
1
Immunoprecipitation of AGO2 in FT3-7 cells
2
Intrathecal Inhibition of CXCL13, STAT3, and NF-κB
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CXCL13 (100 ng/10 μL) and its neutralizing antibody (20 ng/10 μL) were purchased from R&D systems (USA). S3I-201 (STAT3 inhibitor, 10 μg/10 μL) was purchased from Santa Cruz Biotechnology (USA). Ammonium pyrrolidinedithiocarbamate (PDTC, NF-κB inhibitor, 300 μg/5 μL) was purchased from Merck (USA). CXCL13 neutralizing antibody (20 ng/10 μl) was purchased from R&D Systems (USA). IL-6 neutralizing antibody (10 ng/10 μl) was purchased from InvivoGen (USA). Isotype control IgG was purchased from Abcam (UK). The doses of the reagents were chosen according to previous studies [13 (link), 16 (link), 33 (link)–35 (link)]. Reagents were dissolved as following: S3I-201 in 2% DMSO + 98% PBS, PDTC in 20% DMSO + 80% PBS, etanercept in 10% DMSO + 90% PBS, CXCL13 in 0.1% BSA and IL-6 neutralizing antibody in 100% PBS. The drugs and their corresponding vehicle were administrated intrathecally (i.t.) 1 h before behavioral test. Intrathecal injection was performed by a lumbar puncture to deliver to cerebral spinal fluid under isoflurane anesthesia as we previously described [36 (link)].
3
Immunophenotyping of FFA1 in HT-29 Cells
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HT-29 cells cultured in 35 mm dishes were lifted by trypsinization at 37 °C, trypsin activity was stopped by the addition of a complete culture medium, and then cells were pelleted by centrifugation at 600× g for 5 min, and afterwards were fixed by 4% paraformaldehyde for 10 min at 37 °C. Then, they were cooled in ice for 1 min, before being centrifuged at 600× g for 5 min and resuspended using 90% methanol in PBS (137 mM NaCl; 2.7 mM KCl; 10 mM NaHPO4; 2 mM KH2PO4). Cells were then incubated for 30 min at 4 °C, washed twice with PBS, and treated for 60 min with monoclonal anti-FFA1 antibody (rabbit, Abcam, Cambridge, MA, USA) or Isotype control IgG (rabbit, Abcam) at 4 °C. Afterwards, cells were washed twice with PBS and marked with the anti-rabbit Alexa488-bound secondary antibody for 60 min in darkness. Finally, the cells were washed and then resuspended in PBS. Using a FACSCanto II cytometer (BD Biosciences, San Diego, CA, USA), the cells were displayed as plots of forward light scatter versus side light scatter, and the cell population was identified. The mean fluorescence of Alexa488 was determined from a minimum of 10,000 cells using BD FACSDiva 6.1 software (BD Biosciences).
4
ChIP-qPCR analysis of ARNTL binding
5
ChIP-qPCR analysis of ARNTL binding
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Enteric ILC3 from adult C57BL/6J mice were isolated by flow cytometry. Cells were fixed, cross-linked, lysed and chromosomal DNA-protein complex sonicated to generate DNA fragments ranging from 200-400 base pairs as previously described 2 (link). DNA-protein complexes were immunoprecipitated, using LowCell# ChIP kit (Diagenode), with 1μg of antibody against ARNTL (Abcam) and IgG isotype control (Abcam). Immunoprecipitates were uncross-linked and analysed by quantitative PCR using primer pairs flanking ARNTL putative sites (E-boxes) in the Ccr9 locus (determined by computational analysis using TFBS tools and Jaspar 2018). Results were normalized to input intensity and control IgG. Primer sequences were: A: F-CATTTCATAGCTTAGGCTGGCATGG; R-CTAGCTAACTGGTCTCAAAGTCCTC; B: F-GCCTCCCTTGTACTACCTGAAGC; R-TCCCAACACCAGGCCGAGTA; C: F-AGGGTCAATTTCTTAGGGCGACA; R-GCCAAGTGTTCGGTCCCAC; D: F-TCTGGCTTCTCACCATGACCACT; R-TCTAAGGCGTCACCACTGTTCTC, E: F-TTTGGGGAATCATCTTACAGCAGAG; R-ATTCATCCTGGCCCTTTCCTTCTTA; F: F-GCTCCACCTCATAGTTGTCTGG; R-CCATGAGCACGTGGAGAGAAAG; G: F-GGTCGAATACCGCGTGGGTT; R-CCCGGTAGAGGCTGCAAGAAA; H: F-AGGCAAATCTGGGCCTATCC; R-GGCCCAGTACAGAGGGGTCT; I: F-GGCTCAGGCTAGCAGGTCTC; R-TGTTTGGCCAGCATCCTCCA; J: F-ACTCAGAGGTGCTGTGACTCC; R-AGCTTTAGGACCACAATGGGCA.
6
Anti-AAVR Antibody Neutralization Assay
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Wild-type HeLa cells were seeded in 96-well plates at 10,000 cells/well overnight. Anti-AAVR antibody (ab105385) or IgG isotype control (both from Abcam, Cambridge, CA) were incubated with cells (at concentrations ranging from 0.5 to 50 μg/ml in DMEM media) for 1 hr at 4°C. Cells were then infected with AAV2-luciferase at MOI 1,000 vg/cell, and left for 24 hrs at 37°C. A luciferase assay kit (#E1500, Promega, Madison, WI) was used to detect bioluminescence, with measurements being taken on the Promega GLOMAX luminometer. Importantly, the storage buffers of both antibodies did not contain preservatives such as azide that could interfere with the assay. All data presented is representative of two independent experiments.
7
Anti-AAVR Antibody Neutralization Assay
8
Characterization of Porcine cATMSC-Derived Extracellular Vesicles
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Porcine cATMSC-EV were identified by bead-based flow cytometry, screening for EV and MSC markers. EV were covalently coupled to 4-μm aldehyde/sulphate-latex beads (Invitrogen-ThermoFisher Scientific) with a 15 min incubation, and then blocked for 2 h with BCB buffer (PBS, 0.1% BSA, and 0.01% sodium azide (NaN3); Sigma Aldrich). EV-coupled beads were centrifuged at 2000×g for 10 min and re-suspended in BCB buffer. Next, beads were incubated for 30 min at RT with the fluorochrome-conjugated antibodies anti-CD73-PE and anti-CD90-PE-Cy7 (1:50; both from BD); or the primary Ab anti-CD9 (Clone VJ1/20; 1:10), anti-CD63 (Clone TEA3/18; 1:10), anti-CD81 (Clone 5A6; 1:10), anti-CD29 (1:10; BD), anti-CD44 (1:10; AbD Serotec) or IgG isotype control (1:10; Abcam) followed by incubation with the FITC-goat F(ab')2 anti-mouse IgG (1:10; Bionova) or A488-rabbit anti-rat IgG (1:100; AbD Serotec). EV-coupled beads were washed with BCB buffer and spun down at 2000×g for 10 min after each step. Data was acquired in a FACSVerse flow cytometer (BD), and analysed with FlowJo® v10 (BD).
9
Immunohistochemical Analysis of Tissue Samples
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Upon receipt of fresh tissue, a piece was removed and immediately fixed in formalin. The fixed tissue was processed and embedded in paraffin. For histology staining, 5 µm sections were mounted onto glass slides. Rehydration and antigen retrieval were performed using citrate buffer, pH 6.0 (Abcam, Cambridge, UK). Slides were stained with Anti-pan Cytokeratin AE1/AE3 + 5D3 (Abcam, Cambridge, UK) or IgG Isotype control (Abcam, Cambridge, UK) for 2 hours at room temperature. Staining was detected using Mouse and Rabbit Specific HRP/DAB IHC Detection kit (Abcam, Cambridge, UK). Images were taken on an Olympus IX70 microscope with a Jenoptik (Jena, Germany) ProgRes C14plus camera and ProgRes CapturePro software. Brightfield spheroid images were taken on the same microscope with the same camera and software.
10
Cell Adhesion Assay with Peptide Coatings
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The 96-well microplates wells were coated with peptide assemblies (the concentration of FFFIKLLI was kept at 100 μM constantly) for 12 hr at 37 °C before blocked with the Blocking Buffer (2% BSA, 1 mM CaCI2 and 1 mM MnCI2 in PBS) for 1 hr at 37 °C. Cells were collected from culture dishes and suspended in the Blocking Buffer containing anti-laminin-5 (P3H9-2, MAB1947, Chemicon, 5 μg/ml), anti-fibronectin (IST-9, ab6328, Abcam, 20 μg/ml) or IgG isotype control (20 μg/ml, Abcam) before immediately seeded to the coated well (20,000 cells/well) and allowed to incubate at 37 °C for 1 h. The wells were washed for three times with the Blocking Buffer and the phase-contrast images were captured by IncucyteS3 (Essen Bioscience) with a 10× objective.
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