Ibrutinib

Manufactured by Selleck Chemicals

Sourced in United States, Germany, United Kingdom, China

Ibrutinib is a laboratory compound that inhibits a specific enzyme. It is used as a research tool in various scientific studies.

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Lab products found in correlation

163 protocols using ibrutinib

NK Cell-Mediated Killing of CLL Cells

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CLL cells were stained with Cell Trace™ Violet Cell Proliferation Kit (Invitrogen™) then treated for 24-hours with selinexor (Karyopharm Therapeutics), leptomycin B (Sigma), ibrutinib (Selleckchem) or acalabrutinib (Selleckchem). CLL cells were treated with the caspase inhibitor Q-VD-OPh (QVD, 30 µM, Sigma) for 30 min prior to addition of anti-cancer agents. CLL cells were then co-cultured with healthy NK cells at an effector:target (E:T) ratio of either 1:1 or 5:1 for 4 h at 37 °C. After co-culture, cells were stained with propidium iodide (Invitrogen™) and NK cell-specific lysis of CLL cells assessed on a BD FACS Aria II (BD Biosciences) using FACSDiva software (BD Biosciences) and analysed with FlowJo v10.7.1 (BD Biosciences). NK cell-specific Lysis was calculated as follows:

N K s p e c i f i c l y s i s = \frac{(%CLL lysis in coculture) - (%spontaneous CLL lysis)}{Maximum lysis - % spontaneous CLL lysis} * 100

Fisher J.G., Doyle A.D., Graham L.V., Sonar S., Sale B., Henderson I., Del Rio L., Johnson P.W., Landesman Y., Cragg M.S., Forconi F., Walker C.J., Khakoo S.I, & Blunt M.D. (2023). XPO1 inhibition sensitises CLL cells to NK cell mediated cytotoxicity and overcomes HLA-E expression. Leukemia, 37(10), 2036-2049.

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Screening DLBCL Cell Lines with Ibrutinib

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DLBCL cell lines were enumerated and 10,000 cells were seeded in triplicate in a 96-well plate in fresh media. Ibrutinib (Selleck Chem) was dissolved in DMSO and equal volumes of diluted drug were added to cells to reach the indicated final concentration. Cells were cultured with drugs, which were replenished after 48 hours. Metabolic activity was measured at day 4 by adding 10 ul of MTS reagent (Promega) and incubating at 37°C for 4 hours. Absorbance was measured at 490 nm using a 96-well Tecan Infinite 200 Pro plate reader. Absorbance from media-only wells were subtracted and data was normalized to DMSO control unless otherwise stated. GR50 was calculated using the online tool GRcalculator (http://www.grcalculator.org/) ^{30 (link)}. Drug matrix screens and Δbliss calculations were performed as previously described^{31 (link)}.

Phelan J.D., Young R.M., Webster D.E., Roulland S., Wright G.W., Kasbekar M., Shaffer AL I.I.I., Ceribelli M., Wang J.Q., Schmitz R., Nakagawa M., Bachy E., Huang D.W., Ji Y., Chen L., Yang Y., Zhao H., Yu X., Xu W., Palisoc M.M., Valadez R.R., Davies-Hill T., Wilson W.H., Chan W.C., Jaffe E.S., Gascoyne R.D., Campo E., Rosenwald A., Ott G., Delabie J., Rimsza L.M., Rodriguez F.J., Estephan F., Holdhoff M., Kruhlak M.J., Hewitt S.M., Thomas C.J., Pittaluga S., Oellerich T, & Staudt L.M. (2018). A Multiprotein Supercomplex Controlling Oncogenic Signaling in Lymphoma. Nature, 560(7718), 387-391.

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Cytokine Profiling of Ibrutinib-Treated B Cells

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For cytokine profiling, B cells were preincubated with ibrutinib (Selleckchem, Houston, USA) or 0.1% DMSO (control) for 1 h at a concentration of 2 × 10⁶ cells/ml. B cells were then stimulated with the Toll-like receptor 9 agonist CpG (1 μM, ODN2006, Invivogen, San Diego, USA) and cultivated on a 96-well-plate for 24 h at a concentration of 1 × 10⁶/ml. Supernatants were collected and stored at − 20 °C prior to analysis in a multiplex assay (LEGENDplex™ Mix-and-Match-Panel 5-plex, BioLegend, San Diego, USA) according to the manufacturer’s instructions. For data analysis, the LEGENDplex™ Software v8.0 from BioLegend was used.

Einhaus J., Pecher A.C., Asteriti E., Schmid H., Secker K.A., Duerr-Stoerzer S., Keppeler H., Klein R., Schneidawind C., Henes J, & Schneidawind D. (2020). Inhibition of effector B cells by ibrutinib in systemic sclerosis. Arthritis Research & Therapy, 22, 66.

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DLBCL Cell Line Culture Protocol

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DLBCL cell lines were cultured in RPMI 1640 medium (Invitrogen) supplemented with 20 % FBS and 100 U/ml penicillin/streptomycin. OCI-Ly10 cell line was cultured in IMDM medium (Invitrogen) with 20 % human plasma, penicillin/streptomycin and 50 μM β-mercaptoethanol. Jurkat T cells were cultured like DLBCL cells with 10 % FCS. S-Mepazine HCl (Kalexsyn) and Ibrutinib (Selleck Chem) were solved in DMSO and used as indicated. For detection of RelB cleavage fragment proteasome inhibitor MG132 (Merck; 10 μM) was incubated 1h prior to lysis. Antibodies for Western Blot were BCL10 (Abcam EP606Y and Santa Cruz H197), RelB (Cell signaling, C1E4), BCL_XL (Cell Signaling), MALT1 (H300, Santa Cruz), c-FLIP (Alexis Biochemicals) and β-Actin (I-19, Santa Cruz). IL-6 and IL-10 ELISAs (Immunotools) were performed according to the manufacturer's protocols.

Nagel D., Bognar M., Eitelhuber A.C., Kutzner K., Vincendeau M, & Krappmann D. (2015). Combinatorial BTK and MALT1 inhibition augments killing of CD79 mutant diffuse large B cell lymphoma. Oncotarget, 6(39), 42232-42242.

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Antibody Targeting of BTK and XIAP

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Antibodies against BTK (611116) and XIAP (610762) were purchased from BD Transduction Laboratories (San Diego, CA, USA) and anti-CD45 (H-130) antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Mouse monoclonal anti-BTK IgG antibody (clone 53/BTK from BD Biosciences) recognizes human BTK, but does not cross react with ITK (Tec kinase family member). This antibody is also specific for human BTK as neither wildtype nor BTK deficient chicken B cells show any reactivity with this antibody (Douhan Iii et al, 2007 ). Inhibitions studies were always performed with fresh antibodies. Fifty thousand cells were plated per well in a 96-well plate and treated the next day with the appropriate antibody dilution to a final concentration of 1, 5 or 10 µg/ml. Antibodies against β-actin (C-11), caspase 3 (H-277), and PARP-1/2 (H-250) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The commercially available BTK inhibitor, LFM-A13 or α-Cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide, was obtained from Sigma Saint Louis, MO, USA) and prepared as a 10 mM stock solution dissolved in DMSO. Ibrutinib was purchased from Selleckchem, Catalog #S2680.

Guendel I., Iordanskiy S., Sampey G.C., Van Duyne R., Calvert V., Petricoin E., Saifuddin M., Kehn-Hall K, & Kashanchi F. (2015). Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells. Journal of neurovirology, 21(3), 257-275.

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Inhibitor-based Cellular Assays

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GS-5829, the PI3Kδ inhibitor idelalisib, and the SYK inhibitor entospletinib were provided by Gilead Sciences, Inc. (Foster city, CA, USA) as 10 mM solutions in dimethyl sulfoxide (DMSO). JQ1 and the BTK inhibitor ibrutinib were purchased from Selleck Chemicals (Houston, TX, USA).

Kim E., ten Hacken E., Sivina M., Clarke A., Thompson P.A., Jain N., Ferrajoli A., Estrov Z., Keating M.J., Wierda W.G., Bhalla K.N, & Burger J.A. (2019). The BET Inhibitor GS-5829 Targets Chronic Lymphocytic Leukemia Cells and Their Supportive Microenvironment. Leukemia, 34(6), 1588-1598.

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Flow Cytometric Analysis of Apoptosis

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All the chromogen-conjugated antibodies used in flow cytometry and BD Trucount beads were purchased from BD Biosciences. Fluorescein isothiocyanate (FITC)-conjugated annexin V was purchased from Life Technologies. Goat F(ab′)₂ anti-human IgM was purchased from SouthernBiotech. Ibrutinib (PCI-32765) was purchased from Selleck Chemical(12 (link)).

Boysen J., Nelson M., Magzoub G., Maiti G.P., Sinha S., Goswami M., Vesely S.K., Shanafelt T.D., Kay N.E, & Ghosh A.K. (2016). Dynamics of Microvesicle Generation in B Cell Chronic Lymphocytic Leukemia: Implication in Disease Progression. Leukemia, 31(2), 350-360.

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Cytotoxicity Assay for Cancer Cells

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The following drugs used in this study were purchased: ABT‐263 (AdooQ BioScience, Irwin, CA, USA), LY294002 (Cayman Chemical Company, Ann Arbor, MI, USA), Ibrutinib and Idelalisib (Selleck Chemicals, Houston, TX, USA). 293T and SU‐DHL4 (a kind gift from Dr Kunihiko Takeyama, Dana Farber Cancer Institute, Boston, MA, USA) cells were cultured in DMEM (Sigma Aldrich, St Louis, MO, USA) and RPMI (Sigma Aldrich), respectively. Both culture media were supplemented with 10% FCS, 2 mM l‐glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 1 mM sodium pyruvate.

Takagi Y., Shimada K., Shimada S., Sakamoto A., Naoe T., Nakamura S., Hayakawa F., Tomita A, & Kiyoi H. (2016). SPIB is a novel prognostic factor in diffuse large B‐cell lymphoma that mediates apoptosis via the PI3K‐AKT pathway. Cancer Science, 107(9), 1270-1280.

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Preparation of Iron Chelation Agents

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Deferoxamine (Novartis Pharma SAS) was dissolved in sterile distilled water and deferasirox (Selleckchem S1712), was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 300 and 50 mmol/L, respectively. Ironomycin was a kind gift of Raphaël Rodriguez team (11 (link)), this compound was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mmol/L. Other reagents: erastin (Selleckchem S7242, 10 mmol/L in DMSO), ferrostatin-1 (Selleckchem S7243, 50 mmol/L in DMSO), Q-VD Oph (SelleckChem S7311, 10 mmol/L in DMSO, 30′ pretreatment), iron(III) chloride hexahydrate (31232 Sigma Aldrich, 0.1 mol/L in water, 4 hours after treatment), reduced gluthatione GSH (Sigma Aldrich G4251, 0.1 M in PBS) H₂O₂ (Sigma Aldrich 216763), mafosfamide (surrogate of cyclophosphamide, Santa Cruz ChemCruz SC-211761, 10 mmol/L in saline water), gemcitabine (Sellekchem S1149, 50 mmol/L in saline water), doxorubicin (Sellekchem S1208, 20 mmol/L in DMSO), CldU (Abcam ab213715, 20 mmol/L), IdU (Abcam ab142581, 2 mmol/L), 3-methyl adenine (Merck, #189490), β-mercaptoethanol (Sigma, M3148), staurosporine (Selleckchem, S1421), ketokonazole (Sigma, UC280), Avasimibe (from Sigma, PZ0190), ibrutinib (from Sellekchem S2680), entospletinib (from Selleckchem, S7523), venetoclax (from Sellekchem, S8048), AZD-5991 (from Selleckchem, S8643), and A1155463 (from TargetMol, T6748).

Devin J., Cañeque T., Lin Y.L., Mondoulet L., Veyrune J.L., Abouladze M., Garcia De Paco E., Karmous Gadacha O., Cartron G., Pasero P., Bret C., Rodriguez R, & Moreaux J. (2022). Targeting Cellular Iron Homeostasis with Ironomycin in Diffuse Large B-cell Lymphoma. Cancer Research, 82(6), 998-1012.

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Signal Pathway Protein Detection

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Western blots were performed as previously described [9 (link)]. Antibodies for AKT, GSK3, S6, 4EBP1, S6K1, GAPDH, vinculin, and STAT3 were purchased from Cell Signaling Technology. PRAS40 antibody was purchased from Invitrogen. BLNK antibody was purchased from BD Biosciences. GSK690693, MK2206, everolimus, rapamycin, PF-4708671, AT7867, GS-9973, and ibrutinib were purchased from Selleck. LY-333531 was purchased from Tocris. AZD2014, AZD5363, AZD1208, and C₂₁H₂₅ClN₄O₂ were synthesized by AstraZeneca.

Ezell S.A., Wang S., Bihani T., Lai Z., Grosskurth S.E., Tepsuporn S., Davies B.R., Huszar D, & Byth K.F. (2016). Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma. Oncotarget, 7(8), 9163-9174.

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