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Fludarabine

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Fludarabine is a laboratory reagent used for research purposes. It is a purine analog that inhibits DNA synthesis and cell division. The core function of Fludarabine is to serve as a research tool for investigating cellular processes and mechanisms.

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Lab products found in correlation

Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) X vivo 15, by Lonza (2 mentions) Cladribine, by Bio-Techne (2 mentions) Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions) Csu x1, by PerkinElmer (2 mentions) Mouse ifn λ2, by Thermo Fisher Scientific (2 mentions) Human ifn λ1, by Thermo Fisher Scientific (2 mentions) Puromycin, by Merck Group (2 mentions) Recombinant mouse ifn γ, by BioLegend (1 mentions) Mouse ip 10 elisa kit, by Thermo Fisher Scientific (1 mentions) Venetoclax, by MedChemExpress (1 mentions) Daunorubicin, by Selleck Chemicals (1 mentions) Gedatolisib, by Selleck Chemicals (1 mentions) Clofarabine, by Bio-Techne (1 mentions) Volasertib, by Selleck Chemicals (1 mentions) Cytarabine, by Bio-Techne (1 mentions) Decitabine, by Bio-Techne (1 mentions) Ganetespib, by MedChemExpress (1 mentions) Vismodegib, by MedChemExpress (1 mentions) Olaparib, by MedChemExpress (1 mentions)

10 protocols using fludarabine

1

Fludarabine Modulates T Cell Responses

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Naive T cells were incubated with 100 µM fludarabine (Tocris, Bristol) or 0.1 % DMSO (Sigma Aldrich, St. Louis, MO) for 1 h at 37 °C and 5% CO2 in serum-free medium (XVIVO-15, Lonza, Basel). Subsequently, these pretreated or untreated naive T cells were added to IFNγ-pretreated SEB-loaded primary KCs and cocultured for 24 h at 37 °C and 5% CO2. Finally, secreted cytokines were analyzed by the cytokine secretion assay, and STAT phosphorylation was assessed by phospho-flow cytometry.
Orlik C., Deibel D., Küblbeck J., Balta E., Ganskih S., Habicht J., Niesler B., Schröder-Braunstein J., Schäkel K., Wabnitz G, & Samstag Y. (2019). Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin. Cellular and Molecular Immunology, 17(4), 380-394.
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2

IFN-γ-Induced CXCL10 Production

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2 x 104 cells were incubated with Fludarabine (Tocris Bioscience) at 37C for two hours in complete culture media. Then cells were stimulated with 1ng/ml of recombinant mouse IFN-γ (Biolegend) for 24 hours. Mouse CXCL10 protein concentration was measured from supernatants mouse IP-10 ELISA kit (Invitrogen) according to manufacturer’s protocol.
Tan T.G., Zybina Y., McKenna C., Olow A., Rukmini S.J., Wong M.T., Sadekova S., Chackerian A, & Bauché D. (2022). SPATA2 and CYLD inhibit T cell infiltration into colorectal cancer via regulation of IFN-γ/STAT1 axis. Frontiers in Oncology, 12, 1016307.
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3

Evaluation of nucleoside analogs

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CNDAC was purchased from biorbyt (via Biozol, Eching, Germany), 5-azacytidine, cytarabine, cladribine, clofarabine, decitabine, and fludarabine from Tocris Biosciences (via Bio-Techne GmbH, Wiesbaden, Germany), 6-thioguanine, ganetespib, molibresib, olaparib, sapacitabine, venetoclax, and vismodegib from MedChemExpress (via Hycultec, Beutelsbach, Germany), daunorubicin, gedatolisib, and volasertib from Selleckchem (Berlin, Germany), gemcitabine from Hexal (Holzkirchen, Germany), GTP and dATP from Thermo Scientific (Dreieich, Germany), and CNDAC-TP from Jena Bioscience GmbH (Jena, Germany).
Rothenburger T., Thomas D., Schreiber Y., Wratil P.R., Pflantz T., Knecht K., Digianantonio K., Temple J., Schneider C., Baldauf H.M., McLaughlin K.M., Rothweiler F., Bilen B., Farmand S., Bojkova D., Costa R., Ferreirós N., Geisslinger G., Oellerich T., Xiong Y., Keppler O.T., Wass M.N., Michaelis M, & Cinatl J J.r. (2021). Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells. Journal of Experimental & Clinical Cancer Research : CR, 40, 317.
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4

Mitochondrial Dynamics Modulation in Cells

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With exception of Fludarabine, obtained from Tocris Biosciences and CCCP obtained from Sigma, all inhibitors were obtained from Calbiochem. Cells were incubated with 10–50 μM Fludarabine, 2 μM Pyridone 6, 10–50 μM AG490, 10–100 μM 1,2,3,4,5,6-Hexabromocyclohexane (JAK2 inhibitor II, HBC), 5 μM Bay11, 50 μM PDTC, 30 μM PD98059, 100 μM SP600125, 10 μM SB202190 for 30 min, prior to infection or treatment. In case of IFN treatment, cells were incubated in fresh medium with 0.01 μg/ml human IFN-λ1 or mouse IFN-λ2 (Peprotech), 10 U/ml human IFN-β or 50 U/ml mouse IFN-β (PFL Interferon Source) for 2–5h. Keratinocytes were incubated in media containing either 10 μM CCCP or DMSO. Cells were incubated for 48 hours and then infected or incubated with 250 nM MitoTracker Deep Red FM (Molecular Probes) for 30 min at 37°C prior to fixation in 2% paraformaldehyde in PBS. Confocal images were acquired using a spinning disk confocal head (CSU-X1, Perkin Elmer Co., Boston, MA) coupled to a fully-motorized inverted Zeiss Axiovert 200M microscope equipped with a 63X lens (Pan Apochromat, 1.4 NA). The imaging system operates under control of SlideBook 5 (Intelligent Imaging Innovations Inc, Denver, CO). Micrographs were processed with Adobe Photoshop.
Odendall C., Dixit E., Stavru F., Bierne H., Franz K.M., Fiegen A., Boulant S., Gehrke L., Cossart P, & Kagan J.C. (2014). Diverse intracellular pathogens activate Type III Interferon expression from peroxisomes. Nature immunology, 15(8), 717-726.
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5

Preparation of Tryptophan Derivatives and Cytokines

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l-tryptophan, 1-methyl d,l-tryptophan (1-MT) and kynurenine (all from Sigma-Aldrich) were prepared in distilled water. IFN-γ and interleukin (IL)-13 (Peprotech) were resuspended in Iscove's Modified Dulbecco's medium. Bortezomib (Selleck Chemicals), cycloheximide (Calbiochem) and fludarabine (TOCRIS Biosciences) were prepared in dimethylsulfoxide (DMSO). 6-thioguanine, azathioprine and 5-fluorouracil (all from Sigma) were all prepared at 50 mg/ml in NaOH 1M, NH4OH 1M and DMSO, respectively.
Hanafi L.A., Gauchat D., Godin-Ethier J., Possamaï D., Duvignaud J.B., Leclerc D., Grandvaux N, & Lapointe R. (2014). Fludarabine Downregulates Indoleamine 2,3-Dioxygenase in Tumors via a Proteasome-Mediated Degradation Mechanism. PLoS ONE, 9(6), e99211.
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6

Cytokine and Inhibitor Assay Protocol

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Recombinant Human IL-8 (CXCL8, PeproTech, 200–08), Recombinant Human IL-10 (PeproTech, 200–10), Recombinant Human M-CSF (PeproTech, 300–25), Repertaxin (Sigma-Aldrich, 266,359), Fludarabine (Tocris, 3495/10), Sarsasapogenin (Selleck, S3607), Bay11–7082 (Selleck, S2913), Anemoside B4 (Selleck, S9081).
Piao H., Fu L., Wang Y., Liu Y., Wang Y., Meng X., Yang D., Xiao X, & Zhang J. (2022). A positive feedback loop between gastric cancer cells and tumor-associated macrophage induces malignancy progression. Journal of Experimental & Clinical Cancer Research : CR, 41, 174.
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7

Immune Cell Profiling by Flow Cytometry

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For flow cytometry and sorting experiments, antibodies to CD4 (RM4-4), NK1.1 (PK136), CD49b (DX5), CD3 (17A2), Ly6G (1A8), B220 (RA3-6B2), CD11c (N418), CD45.2 (104), CD45.1 (A20), CX3CR1 (SA011F11), IAb (AF6-120.1), CD11b (Mac1), CD90 (G7), lineage markers (17A2/RB6-8C5/RA3-6B2/Ter-119/M1/70), and CD127 (A7R34) were purchased from BioLegend. To stimulate neutrophils in vitro and for in vivo administration, we used either recombinant mouse IFN-λ2 (purchased from Peprotech) or recombinant mouse IFN-λ2 to which polyethylene glycol was attached (provided by Bristol-Myers Squibb). Recombinant mouse IFN-β was purchased from PBL interferonsource. Recombinant human IFN-λ2 and IFN-β were purchased from Peprotech. Puromycin was purchased from Sigma. Where indicated, specific chemical inhibitors were used. To inhibit protein synthesis, cycloheximide (Sigma) was used at a concentration of 10 μg/ml. To inhibit Jak signaling, pyridone 6 (BioVision) was used. To inhibit STAT-1 signaling, fludarabine (Tocris) was used. To inhibit Jak2 signaling, either AG490 (Tocris) or 1,2,3,4,5,6-hexabromocyclohexane (HBC, Tocris) was used. To stimulate neutrophils, either E. coli LPS (Serotype O55:B5 TLR grade, purchased from Enzo) or recombinant mouse TNF (Peprotech) was used.
Broggi A., Tan Y., Granucci F, & Zanoni I. (2017). IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function. Nature immunology, 18(10), 1084-1093.
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8

Mitochondrial Dynamics Modulation in Cells

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With exception of Fludarabine, obtained from Tocris Biosciences and CCCP obtained from Sigma, all inhibitors were obtained from Calbiochem. Cells were incubated with 10–50 μM Fludarabine, 2 μM Pyridone 6, 10–50 μM AG490, 10–100 μM 1,2,3,4,5,6-Hexabromocyclohexane (JAK2 inhibitor II, HBC), 5 μM Bay11, 50 μM PDTC, 30 μM PD98059, 100 μM SP600125, 10 μM SB202190 for 30 min, prior to infection or treatment. In case of IFN treatment, cells were incubated in fresh medium with 0.01 μg/ml human IFN-λ1 or mouse IFN-λ2 (Peprotech), 10 U/ml human IFN-β or 50 U/ml mouse IFN-β (PFL Interferon Source) for 2–5h. Keratinocytes were incubated in media containing either 10 μM CCCP or DMSO. Cells were incubated for 48 hours and then infected or incubated with 250 nM MitoTracker Deep Red FM (Molecular Probes) for 30 min at 37°C prior to fixation in 2% paraformaldehyde in PBS. Confocal images were acquired using a spinning disk confocal head (CSU-X1, Perkin Elmer Co., Boston, MA) coupled to a fully-motorized inverted Zeiss Axiovert 200M microscope equipped with a 63X lens (Pan Apochromat, 1.4 NA). The imaging system operates under control of SlideBook 5 (Intelligent Imaging Innovations Inc, Denver, CO). Micrographs were processed with Adobe Photoshop.
Odendall C., Dixit E., Stavru F., Bierne H., Franz K.M., Fiegen A., Boulant S., Gehrke L., Cossart P, & Kagan J.C. (2014). Diverse intracellular pathogens activate Type III Interferon expression from peroxisomes. Nature immunology, 15(8), 717-726.
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9

Modulation of T Cell Proliferation

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PBTs were incubated with 1 µM Fludarabine (Tocris, Bristol), 1 µM Stattic (Cayman chemical, Ann Abor, Michigan), 10 µM STATV inhibitor (Cayman chemical, Ann Abor, Michigan) or 0.1% DMSO (Sigma Aldrich, St. Louis, Missouri) for 1 h at 37 °C, 5 % CO2 in serum-free medium (XVIVO-15, Lonza, Basel). These cells were washed once in serum-free medium (XVIVO-15, Lonza, Basel), before they were added to IFNγ-pretreated and SEB-loaded primary KCs and were cocultured at 37 °C, 5 % CO2. T cell proliferation was analyzed by destaining of CFDA using flow cytometry after 72 h.
Orlik C., Berschneider K.M., Jahraus B., Niesler B., Balta E., Schäkel K., Schröder-Braunstein J., Souto-Carneiro M.M, & Samstag Y. (2022). Keratinocyte-induced costimulation of human T cells through CD6 - but not CD2 - activates mTOR and prevents oxidative stress. Frontiers in Immunology, 13, 1016112.
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10

Preparing Stock Solutions of Nucleoside Analogs

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Gemcitabine, azacytidine, and decitabine were purchased from LC Labs. Fludarabine, azidothymidine, and cladribine were purchased from Tocris. Cytarabine was purchased from Selleck Chemicals. Puromycin, nucleosides, nucleotides, and dCTP isotope were purchased from Sigma. Isotopes of deoxycytidine, deoxythymidine, deoxyguanosine, and deoxyadensoine were purchased from Cambridge Isotope Laboratories. Deoxyuridine isotope was purchased from Santa Cruz. Cytarabine, decitabine, azidothymidine, and azacytidine were dissolved in DMSO (Sigma) at 50–100mM and stored at −80°C until use. Working stocks were diluted to 5mM in 100% ethanol (VWR) and stored at −20°C until use. Fludarabine and cladribine were dissolved in DMSO at 50mM, then divided to 100 μL aliquots. Aliquots were stored at −80°C. Aliquots were diluted in DMSO to 5mM and divided to 10 μL aliquots and stored at −20°C, to reduce freeze/thaw cycles. Puromycin was dissolved in sterile water at 50mM and stored at −20°C. Gemcitabine was dissolved in DMSO at 10mM and stored at −20°C. nucleosides were dissolved in sterile water at 10mM and stored at −80°C. Nucleoside and nucleotide isotopes were dissolved in HPLC-grade water (Sigma) at 1mM and stored at −80°C.
Dalin S., Sullivan M.R., Lau A.N., Grauman-Boss B., Mueller H.S., Kreidl E., Fenoglio S., Luengo A., Lees J.A., Vander Heiden M.G., Lauffenburger D.A, & Hemann M.T. (2019). Deoxycytidine Release from Pancreatic Stellate Cells Promotes Gemcitabine Resistance. Cancer research, 79(22), 5723-5733.
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