Paclitaxel (LC Laboratories) was initially dissolved in DMSO to get a solution of 0.01 M and subsequently diluted with MCF-7 growth medium to afford stock solutions with a drug concentration in the range of 0.1–50 µM. The concentration of DMSO reached 0.5% (w/v) in the 50 µM Paclitaxel solution and had no significant influence (P > 0.05) on cell viability compared to the untreated cells. In all the remaining working solutions, the concentration of DMSO never exceeded 0.1% (w/v).
Paclitaxel
Manufactured by LC Laboratories
Sourced in United States, Canada
Paclitaxel is a chemically synthesized compound used for research and development purposes. It is a microtubule-stabilizing agent commonly employed in the study of cell biology and cancer research.
Automatically generated - may contain errors
Lab products found in correlation
Cisplatin, by Merck Group (5 mentions)
Erlotinib, by LC Laboratories (5 mentions)
Celltiter blue, by Promega (3 mentions)
Doxorubicin, by LC Laboratories (3 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Paclitaxel, by Polysciences (2 mentions)
Egm 2mv growth media, by Lonza (2 mentions)
Hcaec, by Lonza (2 mentions)
Prism 6.0b, by GraphPad (2 mentions)
Dasatinib, by LC Laboratories (2 mentions)
Staurosporine, by LC Laboratories (2 mentions)
Gemcitabine, by LC Laboratories (2 mentions)
Orlistat, by Merck Group (2 mentions)
Pp242, by Active Motif (2 mentions)
Active bak ab 1, by Cell Signaling Technology (2 mentions)
Rapamycin, by LC Laboratories (2 mentions)
Docetaxel, by LC Laboratories (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Vitamin e, by Merck Group (2 mentions)
57 protocols using paclitaxel
1
Paclitaxel Dissolution and Dilution
2
Preparation of Paclitaxel-Albumin Formulation
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Human serum albumin (lyophilized powder, ≥96%) was obtained from Sigma Aldrich (Oakville, ON, Canada). Paclitaxel (powder) was obtained from LC Laboratories (Woburn, MA, USA). Chloroform and all other chemicals were obtained from VWR International (Mississauga, ON, Canada).
3
Cell Cycle Analysis of Chemotherapeutics
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Cells were seeded at 1 × 105 per well of the 24-well plate and propagated in culture medium. Next day (after 18 hours), culture medium was replaced by the culture medium without drugs (control) or with 100 nM paclitaxel (PCT) or 30 μM adriamycin (LC Laboratories, Woburn, MA). Cells were harvested after 24 hours and fixed in 70% ethanol at 4°C overnight. Fixed cells were washed with phosphate-buffered saline, incubated with 40 μg/mL propidium iodide and 100 μg/mL RNase in phosphate-buffered saline, and cell cycle was analyzed using flow cytometer FACSVersa (Becton, Dickinson and Company, Franklin Lakes, NJ). CYP3A4 and AKR1C2 expression was monitored by qPCR and immunoblotting in parallel samples 48 hours after exposure to drugs.
4
Paclitaxel Delivery to Endothelial Cells
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Example 3
Delivery of paclitaxel to endothelial cells and tissue was studied in vitro using cells grown on Matrigel™ coated cell culture plates. Human coronary endothelial cells (HCAECs, Lonza, Walkersville, Md.) were cultured in EGM™-2MV growth media (Lonza, Walkersville, Md.). One day prior to paclitaxel transfer studies, cells were seed in 96 well BD Matrigel™ Matrix Thin-Layer cell culture plates at 20,000 cells per well in 0.2 mL of medium. Suspensions of paclitaxel (LC Laboratories, Woburn, Mass.) in water were prepared at 11 mg/ml paclitaxel and with PEI (Polysciences, Warrington, Pa.; MW=750 kDa) or PAMAM, ethylene diamine core, gen 4, dendrimer (Sigma, Milwaukee, Wis.; 14,214 Da) at 0.96 mg/mL (92:8 w/w ratio) or iopromide at 11 mg/mL (IOPR, 1:1 w/w ratio). Suspensions were sonicated briefly prior to use. Resulting suspensions (5 μL) were added to the cell culture plates and incubated for 3 or 10 minutes. Suspensions were also added to Matrigel™ coated plates with medium but without cells. After incubation plates were rinsed three times with phosphate buffered saline (200 μL per well) and then allowed to dry overnight. paclitaxel remaining in plates was dissolved in methanol (250 μL) and quantified by HPLC. The amount of transferred paclitaxel is shown in
5
Paclitaxel Formulation for Intravenous Delivery
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Paclitaxel (LC laboratories) was dissolved in 100 % ethanol. Equal volumes of Paclitaxel solution and cremophor (Sigma-Aldrich) were vigorously vortexed for 10 min. Ice-cold saline (80 % of the final volume) was added to freshly made Paclitaxel/cremophor solution immediately before injection. 30 mg/kg Paclitaxel in this solution was injected intravenously via tail vein at 3 times a week for 2 weeks. Survival time after treatment was 1 day.
6
Antiproliferative Effects of Kinase Inhibitors
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Gefitinib (#G-4408), erlotinib (#E-4007), trametinib (#T-8123), crizotinib (#C-7900) and paclitaxel (#P-9600) were purchased from LC Laboratories (Woburn, MA, USA). Cisplatin (#479306) was purchased from Sigma Aldrich (St. Louis, MO, USA). 2D and 3D cultures were incubated in the presence of the drugs for 72 hours. Cell viability was determined using the commercially available alamarBlue® assay kit (Thermo Fisher Scientific, Madison, WI, USA). Apoptosis was analyzed using the Caspase-Glo® 3/7 assay multiplexed with the MultiTox-Fluor GF-AFC life stain (Promega, Madison, WI, USA). Luminescence and fluorescence intensities were measured using the Paradigm detection platform (Molecular Devices, Sunnyvale, CA, USA). Statistical analysis was done using the GraphPad Prism Software 7.03 (GraphPad Software Inc., La Jolla, CA, USA). For the in situ analyses of apoptosis in cocultures the cells were incubated in the presence of 50 nM Gefitinib or 20 nM crizotinib for 24 hours and processed for immunofluorescent microscopy using the cleaved caspase-3 antibody (Cell Signaling Technology, #9661) that specifically detects apoptotic cells.
7
Synergistic Effects of Valproic Acid and Paclitaxel
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Valproic acid (sodium salt, Sigma) was dissolved in sterile water to a stock concentration of 50 mg/ml and stored at −20 °C. Paclitaxel (Lc laboratories) was dissolved in absolute ethanol to 10 mM stock concentration and then diluted to the required concentrations, with complete cell culture medium. The final concentration of ethanol was no greater than 0.1%. This concentration of solvent had no effect on cell viability (measured by MTT assay). Dose–response studies were carried out in order to determine the suitable doses for further experiments. Cell culture treatments were assessed following two different schedules of administration: (i) single drug treatment was performed using 0.5–1–3–6–10–20 mM VPA or 0.01–0.1–1–10–20–50 μM PTX for 24, 48 and 72hs and (ii) dual drug treatment was performed treating cells with different concentrations of VPA for 24 h and then adding PTX at different concentrations for 48 h (Fig. S1).
Supplementary Fig. 1 can be found, in the online version, atdoi:10.1016/j.toxrep.2014.05.005 .
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Supplementary Fig. 1 can be found, in the online version, at
Drug administration schedule. (A) Single drug treatment and (B) combined drug treatment.
8
Evaluating Drug Combination Cytotoxicity
9
MTS Assay for Cell Proliferation Inhibition
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The HCC-1954, HCC-202, AU-565, SKBR-3, BT-474, and T47D cell lines (ATCC) were maintained in RPMI media (Corning) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Corning), whereas MDA-MB-468 and HCC-1419 (ATCC) were cultured in RPMI with 10% FBS, 1% penicillin–streptomycin (HyClone), 1% MEM non-essential amino acids (Gibco), and 100 μM sodium pyruvate (Gibco). The MDA-MB-231 cell line (ATCC) was cultured in DMEM (Corning) with 10% FBS and 1% penicillin–streptomycin (Corning). MTS assays were performed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega) as previously described45 . In brief, 10,000 cells/well were plated into a 96 well plate. Twenty-four hours later, the cells were treated with paclitaxel (LC Laboratories) for an additional 48 h. DMSO and the apoptosis-inducing pan-kinase inhibitor staurosporine (1 μM) were used as negative and positive controls, respectively. The absorbance at 490 nm (OD 490 nm) was normalized against DMSO.
10
Paclitaxel Modulates S. mansoni Infection
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Paclitaxel (LC Laboratories, Woburn, MA, USA) was reconstituted in PBS and a dose
of 25 mg/kg of mice was IP administered one day after intravenous augmentation
of the mice (challenge) with S. mansoni eggs. The dose was
selected as per the prior report.17 (link) Control mice were given PBS only. In the chronic hypoxia model,
Paclitaxel was IP administered at a dose of 25 mg/kg of mice, at days 1 and 8
.
of 25 mg/kg of mice was IP administered one day after intravenous augmentation
of the mice (challenge) with S. mansoni eggs. The dose was
selected as per the prior report.17 (link) Control mice were given PBS only. In the chronic hypoxia model,
Paclitaxel was IP administered at a dose of 25 mg/kg of mice, at days 1 and 8
.
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