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8 protocols using fludarabine

1

Evaluating Inhibitors of Signaling Pathways

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Rottlerin, an inhibitor of PKC (Calbiochem, La Jolla, CA), CID755673, an inhibitor of PKD (Tocris Biosciences, Ellisville, MO), Bay11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenenitrile], an inhibitor of IkB-α phosphorylation (Sigma, St. Louis, MO) and fludarabine, an inhibitor of STAT1 (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the medium at various concentrations between 0 and 100 µM in DMSO (Sigma, St. Louis, MO). DMSO was used as a vehicle control.
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2

Cytokine Signaling Pathway Analysis

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Dulbecco’s modified Eagle medium (DMEM) and Dulbecco’s phosphate buffered saline (DPBS), fatal bovine serum (FBS), TrypLETM Express, pen-strep, glutamax, and protease inhibitor were purchased from Gibco Life Technologies (Grand Island, NY, USA). Human (h) IL-22, hIL-24, and all phosphorylated and secondary antibodies (Section 4.2) were procured from Cell Signaling Technology (Danvers, MA, USA). β-actin was purchased from Novus Biologicals (Littleton, CO, USA). The phosphatase inhibitor PhosSTOP was obtained from Roche (Basel, Switzerland). hIL-20 was purchased from R&D Systems (Minneapolis, MN, USA) and mIL-22 from PeproTech (Rocky Hill, NJ, USA). The specific inhibitor of STAT1 (Fludarabine) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), those of STAT3 and STAT5 from Calbiochem (San Diego, CA, USA), PI3K/Akt inhibitor (LY294002) and MEK1/2-ERK1/2 inhibitor (U0126) from Sigma-Aldrich (St. Louis, MO, USA), and p38 MAP kinase inhibitor (SB203580) from Tocris Bioscience (Bristol, UK).
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3

CLL Cell Isolation and DNA Damage Assay

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Peripheral blood was obtained from CLL patients with written informed consent in accordance with the Declaration of Helsinki and under protocols approved by the Institutional Review Board of the Ohio State University. CLL cells and normal B-cells were isolated using ficoll density gradient centrifugation (Ficoll-Plaque Plus; Amersham Biosciences) and enriched for B-cells using the Rosette-Sep negative selection kit (StemCell Technologies) according to manufacturer protocol. Cryopreserved cells used in the two prognostic datasets were obtained from the CLL Research Consortium (CRC) tissue bank or from the ECOG-2997 clinical trial. Cells were thawed in RPMI 1640 media then washed in PBS to obtain cell pellets.
The OSU-CLL cell line was grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 2mM L-glutamine (Invitrogen), 100U/mL penicillin, and 100ug/mL streptomycin (Sigma). For DNA damage experiments, 2×106 cells were incubated with vehicle (DMSO), 10uM fludarabine (Sigma), 2.36uM mafosfamide (Santa Cruz), or 10uM fludarabine plus 2.36uM mafosfamide. All conditions were given equivalent volumes of DMSO.
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4

Modulating Stat1 and Stat3 in RBC Differentiation

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The Stat1 inhibitor Fludarabine (Santa Cruz Biotechnology, sc-204755)
and the Stat3 inhibitor Stattic (Santa Cruz Biotechnology, sc-202818) were
dissolved in dimethyl sulfoxide (DMSO) at 200 mM and 50 mM, respectively, and
added to the RBC differentiation medium at a final concentration of 1.5
μM.
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5

Measuring Cell Proliferation and Viability

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To measure cell proliferation, cells were seeded in 96-well plates at a density of 1 × 104 cells/well and 1:10 (v/v) Alamar blue (Invitrogen) was added to each well. Fluorescence (excitation 560nm; emission 590nm) was measured 2h after adding Alamar blue and at indicated time points in an Infinite M200 microplate reader (Tecan). To quantify cell viability, cells were seeded in 96-well plates at a density of 1 × 104 cells/well, with or without fulvestrant (Selleck Chemicals), palbociclib (Sigma-Aldrich), ribociclib (Santa Cruz Biotechnology), abemaciclib (Selleck Chemicals), OPG-Fc (Amgen Inc), RANK-Fc (R&D Systems), 3-ATA (Santa Cruz Biotechnology), seliciclib (Focus Biomolecules), and/or fludarabine (Santa Cruz Biotechnology). Complete medium with drugs was replaced every 2 days. After 8 days, viability was measured by Alamar blue assay as described above. For clonogenic assays, cells were seeded in 6-well plates at a density of 2 × 104 - 4 × 104 cells/well for 7 days and allowed to recover for additional 7 days in drug-free medium. Cells were washed and fixed with 4% PFA (Merck) for 10min and stained with 2% crystal violet (Sigma-Aldrich) for 10 min. crystal violet was solubilized with 1% SDS (Merck) for 15–30 min and absorbance at 570 nM was measured in an Infinite M200 microplate reader (Tecan).
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6

Multicolor Flow Cytometry Analysis

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Fluorescent dye-conjugated antibodies that are specific for CD45, CD4, CD8, and Zombie violet were obtained from Biolegend (San Diego, CA). pSTAT1 inhibitor Fludarabine [30 (link)] and pSTAT3 inhibitor Stattic [31 (link)] were obtained from Santa Cruz. The 2W1S and OVA tetramers were provided by the NIH Tetramer Core Facility (Emory University, GA). The cells were stained with 0.15 μl 2W1S and 0.25 μl OVA tetramers. All the antibodies and reagents are listed in Additional file 1 Table S1.
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7

Inhibiting STAT1 and STAT6 Signaling

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A chemical inhibitor of STAT1 (Fludarabine) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), a STAT6 inhibitor (AS1517499) was purchased from Axon Medchem BV (Groningen, The Netherlands), and human recombinant IL-4 and IFN-γ were obtained from BioSource (Camarillo, CA, USA). Phorbol 12-myristate 13-acetate (PMA) and dinitrophenyl-human serum albumin (DNP-HSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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8

Evaluating STAT1 Inhibition on Amoebiasis in Adenocarcinoma and Liver Cancer Cells

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Human adenocarcinoma Caco-2 cells and human liver cancer HepG2 cells from ATCC were grown to confluent monolayers in Dulbecco’s modified Eagle’s medium (DMEM) (catalog number 11995-040; Thermo Fisher) with 10% serum, penicillin, and streptomycin for 3 days (modified from the procedures in references 49 (link) and 50 (link)). Cells grown in 24-well plates to 80 to 90% confluence were washed twice with DPBS, and 500 μl of DMEM medium was added per well. E. histolytica trophozoites (1 × 105 per well) were treated or not with the STAT1 inhibitor fludarabine (50 μM) (catalog number Sc-204755; Santa Cruz) for 30 min prior to IFN-γ (100 ng/ml) stimulation for 20 min. For the interaction, trophozoites were added to each well for 1 h. After incubation, the cells were placed on ice, washed with DPBS, and fixed with 2.5% paraformaldehyde for 10 min. The monolayer was stained with 0.1% methylene blue in 0.1 M borate buffer (pH 8) for 10 min at room temperature. The plates were washed with 0.1 M borate buffer twice to remove excess stain. Finally, 1 ml of 1 N HCl was added to each well for 30 min at 37°C to extract the stain, and the absorbance read on a spectrometer at 655 nm (optical density at 655 nm [OD655]). The percentage of monolayer destruction was calculated as follows: [OD655 (control wells) − OD655 (experimental wells)/OD655 (control wells)] × 100.
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