Log In Sign up
The largest database of trusted experimental protocols

Crt0066101

Manufactured by Bio-Techne
Visit Supplier
Sourced in France, United States

The CRT0066101 is a laboratory equipment product manufactured by Bio-Techne. It is a device designed for use in various scientific and research applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation. As such, the description for this product is not available at this time.

Automatically generated - may contain errors

Lab products found in correlation

Ouabain, by Merck Group (2 mentions) Mcc950, by Merck Group (2 mentions) Nigericin, by Enzo Life Sciences (2 mentions) Cgp084892, by Novartis (2 mentions) Afn700, by Novartis (2 mentions) Baa473, by Novartis (2 mentions) Salmonella typhimurium protein prgl, by Thermo Fisher Scientific (2 mentions) Gramicidin, by Merck Group (2 mentions) Soybean trypsin inhibitor, by Thermo Fisher Scientific (2 mentions) Kb nb 142 70, by Bio-Techne (2 mentions) Rat tail collagen 1, by BD (2 mentions) Anti amylase and anti β actin antibodies, by Merck Group (2 mentions) Anti cytokeratin 19 antibody, by Leica (2 mentions) Anti claudin 18 antibody, by Thermo Fisher Scientific (2 mentions) Anti ras antibody, by Abcam (2 mentions) Anti gst and anti gfp, by Santa Cruz Biotechnology (2 mentions) Anti activated notch1 antibody, by Abcam (2 mentions) Anti pkd2, by OriGene (2 mentions) Anti pkd3 antibody, by Fortis Life Sciences (2 mentions) Anti ps744 748 pkd antibodies, by Cell Signaling Technology (2 mentions)

11 protocols using crt0066101

1

Cytokine/Chemokine Response in Placental Macrophages

Check if the same lab product or an alternative is used in the 5 most similar protocols
Placental macrophages were cultured in 6-well plates using a density of 4×106 macrophages per well in 2mL RPMI 1640 supplemented with 10% charcoal-stripped, dextran treated, heat inactivated FBS at 37ºC with 5% carbon dioxide. Following 1 hr pretreatment with CRT0066101 (CRT, Tocris Bioscience; Ellisville, MO) or vehicle and subsequent removal of CRT0066101, cells were infected with GBS (0.1 bacterium per macrophage) for 6 hours, and supernatants collected. A custom 15-plex Millipore MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay was run according to instructions provided by the manufacturer. Briefly, 25 μL of supernatant or standard/quality control was loaded into a 96-well plate followed by addition of assay buffer, matrix solution, and magnetic beads and allowed to incubate overnight at 4°C. Wells were then washed and incubated for 1hr with detection antibodies followed by incubation with Streptavidin-Phycoerythrin for 30 min. Wells were then washed and the plate was analyzed using x-map technology on the Luminex® MagPix® machine. Results were graphed and analyzed using GraphPad Prism® (GraphPad Software, La Jolla, CA).
Sutton J.A., Rogers L.M., Dixon B., Kirk L., Doster R., Algood H.M., Gaddy J.A., Flaherty R., Manning S.D, & Aronoff DM D.R. (2019). Protein Kinase D Mediates Inflammatory Responses of Human Placental Macrophages to Group B Streptococcus. American journal of reproductive immunology (New York, N.Y. : 1989), 81(3), e13075.
+ Open protocol
+ Expand
2

Activation of Inflammasome Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols
Nigericin (Enzo, BML-CA421-005), ouabain (Sigma #O3125), gramicidin (Sigma #G5002), niclosamide (Adipogen #AG-CR1-3643), MSU crystals (InvivoGen # tlrl-msu), DMSO, used as vehicle for compound dilutions (Sigma #D2650), MCC950 (either from Sigma, #5381200001 or synthesized at Novartis), CGP084892 and AFN700 (both synthesized at Novartis), CRT0066101 (Tocris #4975). The pyrin activating compound BAA473 has been previously described [16 (link)]; it was synthesized at Novartis. The NLRC4 activating recombinant Salmonella Typhimurium Protein Prgl was from BioSource #MBS1177087.
Heiser D., Rubert J., Unterreiner A., Maurer C., Kamke M., Bodendorf U., Farady C.J., Roediger B, & Bornancin F. (2021). Evaluation of protein kinase D auto-phosphorylation as biomarker for NLRP3 inflammasome activation. PLoS ONE, 16(11), e0248668.
+ Open protocol
+ Expand
3

Antibody Source and Cell Culture Reagents

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
The anti-amylase and anti-β-actin antibodies were from Sigma-Aldrich (St Louis, MO), anti-cytokeratin-19 antibody from Leica Microsystems (Buffalo Grove, IL), anti-claudin-18 antibody from Invitrogen (Carlsbad, CA), anti-Ras antibody from Epitomics (Burlingame, CA), anti-GST and anti-GFP from Santa Cruz (Santa Cruz, CA), anti-activated Notch1 antibody from Abcam (Cambridge, MA), anti-PKD1 from Antibodies-online Inc. (Atlanta, GA), anti-PKD2 from Acris Antibodies (San Diego, CA), anti-PKD3 antibody from Bethyl Laboratories (Montgometry, TX), and anti-pS744/748-PKD antibodies from Cell Signaling Technology (Danvers, MA) or Abcam. Recombinant human TGFα was purchased from Chemicon (Billerica, MA) and R&D Systems (Minneapolis, MN). Dexamethasone was from Sigma-Aldrich, soybean trypsin inhibitor and collagenase I from Affymetrix (Santa Clara, CA). Phenol red-free Matrigel and rat tail collagen I were from BD Biosciences (San Diego, CA). Hoechst 33342 was purchased from Invitrogen (Grand Island, NY). The PKD inhibitor CRT0066101 was from TOCRIS (Ellisville, MO) and kb-NB-142-70 was described previously 51 (link). The γ-secretase inhibitors DAPT and R04929097 were from Cellagen Technology (San Diego, CA).
Liou G.Y., Döppler H., Braun U.B., Panayiotou R., Buzhardt M.S., Radisky D.C., Crawford H.C., Fields A.P., Murray N.R., Wang Q.J., Leitges M, & Storz P. (2015). Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia. Nature communications, 6, 6200.
+ Open protocol
+ Expand
4

Antibody Source and Cell Culture Reagents

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
The anti-amylase and anti-β-actin antibodies were from Sigma-Aldrich (St Louis, MO), anti-cytokeratin-19 antibody from Leica Microsystems (Buffalo Grove, IL), anti-claudin-18 antibody from Invitrogen (Carlsbad, CA), anti-Ras antibody from Epitomics (Burlingame, CA), anti-GST and anti-GFP from Santa Cruz (Santa Cruz, CA), anti-activated Notch1 antibody from Abcam (Cambridge, MA), anti-PKD1 from Antibodies-online Inc. (Atlanta, GA), anti-PKD2 from Acris Antibodies (San Diego, CA), anti-PKD3 antibody from Bethyl Laboratories (Montgometry, TX), and anti-pS744/748-PKD antibodies from Cell Signaling Technology (Danvers, MA) or Abcam. Recombinant human TGFα was purchased from Chemicon (Billerica, MA) and R&D Systems (Minneapolis, MN). Dexamethasone was from Sigma-Aldrich, soybean trypsin inhibitor and collagenase I from Affymetrix (Santa Clara, CA). Phenol red-free Matrigel and rat tail collagen I were from BD Biosciences (San Diego, CA). Hoechst 33342 was purchased from Invitrogen (Grand Island, NY). The PKD inhibitor CRT0066101 was from TOCRIS (Ellisville, MO) and kb-NB-142-70 was described previously 51 (link). The γ-secretase inhibitors DAPT and R04929097 were from Cellagen Technology (San Diego, CA).
Liou G.Y., Döppler H., Braun U.B., Panayiotou R., Buzhardt M.S., Radisky D.C., Crawford H.C., Fields A.P., Murray N.R., Wang Q.J., Leitges M, & Storz P. (2015). Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia. Nature communications, 6, 6200.
+ Open protocol
+ Expand
5

Inflammation Modulation Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
The following reagents were used: polyethylenimine (PEI; Sigma-Aldrich), polybrene (Santa-Cruz), PMA (Invivogen), doxycycline (Sigma-Aldrich), LPS (O111:B4; Sigma-Aldrich), nigericin (Invivogen), ATP (Sigma-Aldrich), MCC950 (Adipogen), CY-09 (Tocris), G5 (Calbiochem), CRT0066101 (Tocris), PI (Immunochemistry Technologies), Hoechst (Immunochemistry Technologies).
Cosson C., Riou R., Patoli D., Niu T., Rey A., Groslambert M., De Rosny C., Chatre E., Allatif O., Henry T., Venet F., Milhavet F., Boursier G., Belot A., Jamilloux Y., Merlin E., Duquesne A., Grateau G., Savey L., Jacques Maria A.T., Pagnier A., Poutrel S., Lambotte O., Mallebranche C., Ardois S., Richer O., Lemelle I., Rieux-Laucat F., Bader-Meunier B., Amoura Z., Melki I., Cuisset L., Touitou I., Geyer M., Georgin-Lavialle S, & Py B.F. (2024). Functional diversity of NLRP3 gain-of-function mutants associated with CAPS autoinflammation. The Journal of Experimental Medicine, 221(5), e20231200.
+ Open protocol
+ Expand
6

Placental Macrophage Infection Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Placental macrophages were seeded at a density of 4×106 cells per well in a polystyrene, 6-well culture plate in RPMI 1640 (Gibco) supplemented with 10% charcoal-stripped, dextran treated heat inactivated FBS at 37ºC with 5% carbon dioxide overnight (~16 h). Before treatment, cells were visually inspected to verify proper morphology. Cells were pretreated with 5 μM PKD inhibitor CRT0066101 (CRT, Tocris Bioscience; Ellisville, MO) or vehicle for 1hr prior to infection, after which time the inhibitor was removed. Cells were then infected with GBS (MOI of 0.1:1 or 10:1) following CRT pretreatment for indicated time points at 37ºC with 5% carbon dioxide. Cells and supernatants were collected for downstream analysis.
Sutton J.A., Rogers L.M., Dixon B., Kirk L., Doster R., Algood H.M., Gaddy J.A., Flaherty R., Manning S.D, & Aronoff DM D.R. (2019). Protein Kinase D Mediates Inflammatory Responses of Human Placental Macrophages to Group B Streptococcus. American journal of reproductive immunology (New York, N.Y. : 1989), 81(3), e13075.
+ Open protocol
+ Expand
7

Kinase Assay for PAH Phosphorylation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Recombinant GST-PAH (vector synthesized by Vectorbuilder) was produced in Escherichia coli (BL21) as GST fusion protein, and purified by affinity chromatography on glutathione–Sepharose columns. Recombinant human proteins, PKD3 and PKA, were both purchased from Enzo biosciences and SignalChem Biotech, respectively. Kinase reactions were performed in reaction buffer (Cell Signaling Technology) in the presence of cold (nonradioactive) ATP (Cell Signaling Technology) for 30 min at 30°C. As indicated in the experiment, 1 μM of CRT0066101 (Tocris) was added to the corresponding condition. Proteins from the kinase reactions were boiled in 5× Laemmli buffer and analyzed by Western blotting. Membrane was incubated with the appropriate primary antibody against the phosphorylated motif (RxxS/T*) (Cell Signaling Technology), PAH (proteintech, PK), and PKD3 (Cell signaling Technology).
Loza-Valdes A., Mayer A.E., Kassouf T., Trujillo-Viera J., Schmitz W., Dziaczkowski F., Leitges M., Schlosser A, & Sumara G. (2021). A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism. Life Science Alliance, 4(8), e202000863.
+ Open protocol
+ Expand
8

Generation and Characterization of MSU Crystals

Check if the same lab product or an alternative is used in the 5 most similar protocols
MSU crystals were generated as described 12 (link). Briefly, we dissolved 1,68g of uric acid (Sigma-Aldrich) into 500mL of water containing 0,01M of NaOH by heating at 70°C, pH was adjusted to 7,1-7,2. Then, the uric acid solution was left at room temperature until the crystals formed under mild agitation. Crystals were then washed in ethanol, dried, weighted and thermally treated (250°C for an hour) and finally sonicated to obtain crystals <50µm in length. They were aliquoted in sterile PBS and frozen at -20°C until use. Poly-(dA:dT) and Lipofectamine 3000 were purchased from Invivogen and Invitrogen, respectively; LPS (Salmonella abortus equi), ATP and Colchicine from Sigma-Aldrich; ELISA kits (IL-1β, IL-6, TNFα, MPO) from Biotechne. Thioglycolate was home-made from brewer's thioglycolate (BD). The various antibodies used for Western blot were from Biotechne (anti-mouse IL-1β, AF401, and secondary HAF-109) or from Abcam (anti-tubulin) and Sigma-Aldrich (anti-vinculin). Anti-Gr1, Anti-CD11b, anti-CD14 and 7-AAD were obtained from eBioscience. CRT0066101 was provided from the lab of Romeo Ricci and is available from Tocris Bioscience; Anakinra was generously provided by Ommar S. Omarjee and Alexandre Belot (Lyon University, France) and Etanercept by Christian Von Frenckel (Liege University, Belgium).
Mariotte A., De Cauwer A., Po C., Abou-Faycal C., Pichot A., Paul N., Aouadi I., Carapito R., Frisch B., Macquin C., Chatelus E., Sibilia J., Armspach J.P., Bahram S, & Georgel P. (2020). A mouse model of MSU-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1β as relevant therapy for gout patients. Theranostics, 10(5), 2158-2171.
+ Open protocol
+ Expand
9

Characterization of PKD Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols
CCK was from American Peptide (Sunnyvale, CA); Medium 199 was from GIBCO (Grand Island, NY). ATP and [γ-32P] ATP were from Perkin Elmer (Torrance, CA). CRT0066101 and CID755673 were obtained from TOCRIS (Mo, USA). Nitrocellulose membranes were from Schleicher and Schuell BioSience. Carbachol and GF1 (also known as GF109203X or bisindolylmaleimide I) were from Calbiochem (La Jolla, CA). Antibodies against PKD C-20, IκB-α, or LDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphoserine 744/748 PKD antibody that detects primarily the phosphorylated state of Ser 744 (Jacamo et al., 2008 (link)), phosphoserine 916 PKD antibody, antibodies for NF-κB P65, phosphoserine 32/36 IκB-α, GAPDH, ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). IL-6 antibody was from PeproTech (Rocky Hill, NJ) and MCP-1 antibody was from Antibodies-Online Inc. (Secaucus, NJ). Protein-A-agarose was from Roche Applied Science (Mannheim, Germany) and PKD substrate syntide-2, was from Bachem (Chicago, IL). Other items were from standard suppliers or as indicated in text.
Yuan J., Tan T., Geng M., Tan G., Chheda C, & Pandol S.J. (2017). Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis. Frontiers in Physiology, 8, 1014.
+ Open protocol
+ Expand
10

Oligodeoxynucleotide-mediated Immune Modulation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Nuclease-resistant phosphorothioate oligodeoxynucleotides (S-ODN) 1826 (CpG DNA) were purchased from Coley Pharmaceutical Group (Kanata, ON, Canada) and further purified by ethanol precipitation. S-ODN had no detectable endotoxins by Limulus assay. The sequences of S-ODN used have been previously reported (41 (link)). Peptidoglycan (PGN) was purchased from Invivogen (San Diego, CA). PMA, ionomycin, and D-galactosamine (D-GalN) were purchased from Sigma Chemical Co. (St. Louis, MO). Gö6976, Gö6983, and CRT0066101 were purchased from Tocris (Minneapolis, MN). IRAK inhibitor 1 (IRAK4 inhibitor; 6-imidazo[1,2-a]pyridin-3-yl-N-piperidin-4-ylpyridin-2-amine) was purchased from APExBIO (Houston, TX).
Upadhyay K., Park J.E., Yoon T.W., Halder P., Kim Y.I., Metcalfe V., Talati A.J., English B.K, & Yi A.K. (2017). Group B streptococci induce proinflammatory responses via a protein kinase D1-dependent pathway. Journal of immunology (Baltimore, Md. : 1950), 198(11), 4448-4457.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!