Palbociclib

Manufactured by Merck Group

Sourced in United States, Germany, France

Palbociclib is a cyclin-dependent kinase (CDK) 4/6 inhibitor, a type of laboratory equipment used for cell cycle regulation research. It functions by selectively inhibiting the activity of CDK4 and CDK6, which are involved in the control of cell cycle progression.

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Lab products found in correlation

Nocodazole, by Merck Group (4 mentions) Abemaciclib, by MedChemExpress (4 mentions) Doxorubicin, by Merck Group (3 mentions) Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Paclitaxel, by Merck Group (2 mentions) Ribociclib, by Selleck Chemicals (2 mentions) Hydroxyurea hu, by Fujifilm (2 mentions) Abemaciclib, by Merck Group (2 mentions) Hlm006474, by Merck Group (2 mentions) Abemaciclib, by Selleck Chemicals (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Alpelisib, by MedChemExpress (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) Mg132, by Bio-Techne (2 mentions) Everolimus, by Merck Group (2 mentions) Ribociclib, by MedChemExpress (2 mentions) Nu7441, by Selleck Chemicals (2 mentions) Crystal violet, by Merck Group (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Fluorouracil, by Merck Group (1 mentions)

51 protocols using palbociclib

Isolating and Culturing Synoviocytes for Cytokine and Therapeutic Response Analysis

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Synoviocytes were isolated from synovial tissue by using standard procedures (19 ). Synoviocytes from patients with RA were digested with Liberase TM Research Grade (Sigma–Aldrich) for 90 min at 37°C. The digested synoviocyte slurries were filtered through a 100 μm cell strainer (EASYstrainer™ Cell; Greiner Bio-One, Kremsmünster, Austria). Cells were suspended in the growth medium consisting of Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated at 37°C in 5% CO₂. Cells at passages 3 to 6 were used in all experiments.
For cytokine stimulation, cells were treated with or without various concentrations (1, 10, and 100 ng/mL) of PDGF-BB (BioLegend; San Diego, CA, USA), TGF-β (BioLegend), or TNF-α (BioLegend) for 2 days, as per our approved experimental design. The role of therapeutic drugs was determined by treating cells stimulated with cytokines with 10, 25, or 50 μg/mL etanercept (Pfizer, NY, USA) or 7.5 μM or 15 μM palbociclib (Sigma–Aldrich), or a combination of 25 μg/mL etanercept and 7.5 μM palbociclib for 1 day, as per our experimental design. These doses of cytokines and inhibitors were selected based on the findings of previous studies (20 (link)–23 (link)). WST-8 assays were performed to assess cell proliferation by using the Cell Counting Kit-8 (CK04; Dojindo).

Matsumura T., Saito Y., Suzuki T., Teramoto A., Ozasa Y., Yamashita T., Fujimiya M, & Saito-Chikenji T. (2019). Phosphorylated Platelet-Derived Growth Factor Receptor-Positive Cells With Anti-apoptotic Properties Accumulate in the Synovium of Patients With Rheumatoid Arthritis. Frontiers in Immunology, 10, 241.

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Palbociclib Dosage and Administration

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Palbociclib was obtained from Pfizer Inc. Palbociclib powder was stored at room temperature and protected from light.
For in vitro experiments, a 2.5 mM stock of Palbociclib was prepared in DMSO and stored as single use aliquots at -80ºC.
For in vivo experiments, Palbociclib was dissolved in Sodium L-Lactate (Sigma-Aldrich; St. Louis, MO) buffer (50mM, pH 4.0). Cre:ER T+/-PTEN f/f mice were given a single daily dose of 100 mg/kg of Palbociclib by oral gavage, starting two weeks after tamoxifen injection. For Palbociclib acute treatment, Cre-ER T+/-PTEN fl/fl animals were treated for three consecutive days with 75, 100 or 150 mg/kg of the inhibitor. For survival experiments, mice received 5 doses per week until the moment of sacrifice. In each experiment, control animals were given vehicle following the same schemes.

Dosil M.A., Mirantes C., Eritja N., Felip I., Navaridas R., Gatius S., Santacana M., Colàs E., Moiola C., Schoenenberger J.A., Encinas M., Garí E., Matias-Guiu X, & Dolcet X. (2017). Palbociclib has antitumour effects on Pten-deficient endometrial neoplasias. The Journal of pathology, 242(2).

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Palbociclib-Resistant Breast Cancer Cell Lines

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MCF7 and T47D cells were purchased from the American Type Cell Collection (Manassas, VA, USA). Palbociclib-resistant sublines were established via continuous exposure to a constant concentration of Palbociclib for more than 6 months as described in the Supplementary Methods. Three representative clones (MCF7-P1, MCF7-P2, and T47D-PR) were used in this study. Eribulin was provided by Eisai Co., Ltd. (Tokyo, Japan). Abemaciclib, dinaciclib, and everolimus were purchased from Selleck Biotech Co., Ltd. (Tokyo, Japan). Palbociclib, paclitaxel, doxorubicin, and fluorouracil were purchased from Sigma-Aldrich (Saint Louis, MO, USA). This study was approved by the Medical Ethics Committee on Clinical Investigation of Shinshu University (No. 341).

Ono M., Oba T., Shibata T, & Ito K.I. (2021). The mechanisms involved in the resistance of estrogen receptor-positive breast cancer cells to palbociclib are multiple and change over time. Journal of Cancer Research and Clinical Oncology, 147(11), 3211-3224.

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Cell Line Characterization and Inhibitor Screening

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LNCaP cell line was obtained from ATCC, the v-Src PEC and the NeuT-PEC cell lines were previously described (18 (link)). Original cells were expanded and stored in the liquid nitrogen at early passage. During the experiments, the morphology of all cell lines was checked under phase contrast microscope routinely. For LNCaP cell line, proliferation and AR abundance in response to DHT stimulation were tested by MTT assay and Western-blot. For v-Src-PEC and NeuT-PEC cell lines, the proliferation in response to Src kinase inhibitor or NeuT inhibitor was tested. V-Src or NeuT expression in these cells was checked by Western-blot for verification. All of the newly revived cells were treated with BM-cyclins (Roche) and the mycoplasma contamination was determined with Hoechst 33258 staining under high magnification fluorescent microscope routinely. DNA transfection and luciferase assays were performed as previously described (1 (link),18 (link)). The CBF-Luc and -3,400 cyclin D1-Luc reporter plasmids were previously described (19 (link),20 (link)). The Src kinase inhibitor PP1 (4-amino-5-(4-methylphenyl)-7-(t-butel)pyrazolo-d-3-4-pyrimidine (Calbio Chem) and Dasatinib (BMS-354825, Selleckchem), the CDK inhibitor Abemaciclib (MedChem Express), Palbociclib (Sigma-Aldrich), Ribociclib (Selleckchem) and the EGFR inhibitor Canertinib (Selleckchem) were used at the indicated doses.

Ju X., Jiao X., Ertel A., Casimiro M.C., Di Sante G., Deng S., Li Z., Di Rocco A., Zhan T., Hawkins A., Stoyanova T., Ando S., Fatatis A., Lisanti M.P., Gomella L.G., Languino L.R, & Pestell R.G. (2016). SRC ONCOGENE INDUCES TROP2 PROTEOLYTIC ACTIVATION VIA CYCLIN D1. Cancer research, 76(22), 6723-6734.

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Palbociclib Inhibits Retinal Proliferation

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Cdk4/6 inhibition in mice was performed by resuspending Palbociclib (Sigma-Aldrich Cat# PZ0383) in 50 mM Sodium lactate (Sigma Cat# L7022) at 12 mg/mL, then administered to pups at P3, P4, and P5 by oral gavage. Retinas were isolated at P6. EdU incorporation assay was performed using the Click-It EdU Cell Proliferation Kit (ThermoFisher Cat# C10340) per manufacturer’s instructions, with EdU injection into mice 5 h before euthanasia at P6.

Chavkin N.W., Genet G., Poulet M., Jeffery E.D., Marziano C., Genet N., Vasavada H., Nelson E.A., Acharya B.R., Kour A., Aragon J., McDonnell S.P., Huba M., Sheynkman G.M., Walsh K, & Hirschi K.K. (2022). Endothelial cell cycle state determines propensity for arterial-venous fate. Nature Communications, 13, 5891.

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Modulating Cell Cycle Progression in T Cells

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All cell cycle inhibitors were titrated on CFSE labeled CD8 T cells to identify a dose that inhibited proliferation by approximately 50%. Mouse and human CD8 T cells showed similar responses to each drug; therefore, the same concentrations were used for both species. Palbociclib (PD 0332991, Sigma product #PZ0383) was used at 500nM. Abemaciclib (LY2835219, obtained from Eli Lilly Pharmaceuticals) was used at 100 nM. Ribociclib (LEE011, Selleckchem product #S7440) was used at 200 nM. CDK1/2i (RO-3306, Selleckchem product #S7747) was used at 4.5 μM. Aurora kinase inhibitor (MLN-8054, Selleckchem product #S1100) was used at 1 μM. Polo-like kinase inhibitor (GSK-461364, Selleckchem product #S2193) was used at 0.15 μM. CDK7i (YKL-5124) was developed by Nathanael Gray’s lab as reported (53 (link)) and used at 100 nM. CDK4/6 degrader (BJS 2–162) was developed by Nathanael Gray’s lab as reported (32 (link)) and used at 2 μM.

Heckler M., Ali L.R., Clancy-Thompson E., Qiang L., Ventre K.S., Lenehan P., Roehle K., Luoma A., Boelaars K., Peters V., McCreary J., Boschert T., Wang E.S., Suo S., Marangoni F., Mempel T.R., Long H.W., Wucherpfennig K.W., Dougan M., Gray N.S., Yuan G.C., Goel S., Tolaney S.M, & Dougan S.K. (2021). Inhibition of CDK4/6 promotes CD8 T cell memory formation. Cancer discovery, 11(10), 2564-2581.

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Palbociclib inhibits A673 cell growth

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A673 cells were treated with Palbociclib (PD 0332991 ISETHIONATE, Sigma-Aldrich) at a final concentration of 1.5 uM for 24 hr.

Boulay G., Sandoval G.J., Riggi N., Iyer S., Buisson R., Naigles B., Awad M.E., Rengarajan S., Volorio A., McBride M.J., Broye L.C., Zou L., Stamenkovic I., Kadoch C, & Rivera M.N. (2017). Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain. Cell, 171(1), 163-178.e19.

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Cell Culture Assay Compound Solubilization

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All drugs were solubilized in 100% DMSO for a stock concentration of 10mM for use in all cell culture assays. Palbociclib (Sigma #PZ0199), ribociclib (Med Chem Express #HY-15777A), abemaciclib (Med Chem Express #HY-16297A), staurosporin (Sigma #S6942), NU7441 (Selleck #S2638), and AZD7762 (Sigma #SML0350) were all purchased commercially.

Pesch A.M., Hirsh N.H., Chandler B.C., Michmerhuizen A.R., Ritter C.L., Androsiglio M.P., Wilder-Romans K., Liu M., Gersch C.L., Larios J.M., Pierce L.J., Rae J.M, & Speers C.W. (2020). Short Term CDK4/6 Inhibition Radiosensitizes Estrogen Receptor Positive Breast Cancers. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(24), 6568-6580.

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Cell Synchronization Using Palbociclib and Hydroxyurea

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To synchronize hTERT-RPE1 cells at the G1 phase, palbociclib (Sigma-Aldrich) was used as previously described (32 (link),33 (link)) with modifications. Cells were plated at 1.3 × 10⁴ cells/cm², incubated for 6 h, and treated with 1 μM or 150 nM palbociclib for 18 h. To release the cells from the arrest, the cells that had been treated with 150 nM palbociclib were washed three times with pre-warmed culture medium, and then cultured until collection at the indicated times. To synchronize hTERT-RPE1 cells in the S phase, the cells were plated and incubated as described above, and then treated with 200 μM hydroxyurea (HU) (Fujifilm) (34 (link)) for 18 h.

Hayashi-Takanaka Y., Hayashi Y., Hirano Y., Miyawaki-Kuwakado A., Ohkawa Y., Obuse C., Kimura H., Haraguchi T, & Hiraoka Y. (2021). Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry. Nucleic Acids Research, 49(21), 12152-12166.

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Palbociclib and Radiation Effects

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Cells were treated with increasing doses of single agent palbociclib (PZ0383) (Sigma-Aldrich, St Louis, MO, USA). Treated cell viability was normalized to untreated control cells. Combination palbociclib and radiation treatment was also evaluated, cells were treated with palbociclib 1hr prior to single-fraction 2 or 4 Gy radiation treatment. Five days after treatment cell viability was quantified using alamarBlue cell viability reagent according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA).

Ruiz F.J., Sundaresan A., Zhang J., Pedamallu C.S., Halle M.K., Srinivasasainagendra V., Zhang J., Muhammad N., Stanley J., Markovina S., Tiwari H.K., Grigsby P.W., Krakstad C., Schwarz J.K, & Ojesina A.I. (2021). Genomic Characterization and Therapeutic Targeting of HPV Undetected Cervical Carcinomas. Cancers, 13(18), 4551.

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