HPLC was performed to estimate taxol production in the sample extracts. For HPLC analysis, the sample extracts were diluted in the mobile phase and subjected to HPLC (Perkin Elmer, USA), performed using 200 Ic pump (Perkin Elmer) equipped with reverse phase C18 5 μm column (Merck, LiChrosolv) and 785A Absorbance Detector (Applied Biosystem) [16 (link)]. Briefly, extracted test samples (crude taxol) were filtered through a 0.2 μm filter. The mobile phase consisted of methanol: water, 80:20 (v/v). 10 μL of the crude sample was injected each time with 1 mL per min flow rate and was detected at 227 nm [25 (link)]. NetWin Software (Netel Chromatographs, India) was used to monitor absorbance at 227 nm. Taxol presence was verified by comparing the retention time of the test samples with that of the standard taxol (Paclitaxel, M.P Biomedicals).
Paclitaxel
Manufactured by MP Biomedicals
Sourced in United States, France, United Kingdom
Paclitaxel is a natural product isolated from the bark of the Pacific yew tree. It is a microtubule-stabilizing agent that inhibits cell division by promoting the assembly of microtubules and preventing their disassembly.
Automatically generated - may contain errors
Lab products found in correlation
Doxorubicin, by MP Biomedicals (2 mentions)
Verapamil, by MP Biomedicals (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by MP Biomedicals (1 mentions)
N acetyl l cysteine nac, by Beyotime (1 mentions)
Mg132, by MedChemExpress (1 mentions)
Cisplatin, by Bio-Techne (1 mentions)
Doxorubicin, by Bio-Techne (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by American Type Culture Collection (1 mentions)
Streptomycin, by American Type Culture Collection (1 mentions)
Crl 2190, by American Type Culture Collection (1 mentions)
Ccl 34, by American Type Culture Collection (1 mentions)
Pac 1, by Bio-Techne (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Greiner (1 mentions)
5 protocols using paclitaxel
1
Taxol Production Estimation using HPLC
2
Cytotoxicity Assay for Drug Resistance
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Paclitaxel, doxorubicin, verapamil, and MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from MP Biomedicals (Santa Ana, CA). P-gp inhibitors identified though in silico screening were purchased in small quantities through SIA MolPort (Riga, Latvia). Stock solutions (10–100 mmol/L) of all drugs and experimental compounds were prepared in DMSO and stored as aliquots at −20°C. On the day of the experiment, working solutions of compounds prepared in DMSO were further diluted in culture media such that the final DMSO concentration was ≤1% (v/v). MTT solution was prepared as 5 mg/mL in phosphate buffered saline (PBS; 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, 1.8 mmol/L KH2PO4) and sterile filtered through 0.22 μm nylon filter before use. Cell culture materials were purchased from Corning Inc. (Corning, NY) unless otherwise stated. GraphPad Prism∼ version 6.05 for Windows was used for plotting data and IC50 values were calculated from nonlinear, four-parameter logistic curve fitting (GraphPad Software, La Jolla, CA).
3
Extraction and Analysis of Paclitaxel from Fungal Isolate
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Erlenmeyer flask containing 100 mL modified S7 liquid medium [16 (link)] was seeded with 4.79 × 104 spores per mL of the fungal isolate TPF-06 and incubated at 25 ± 1 °C for 21 days with agitation speed at 150 rpm in an incubator shaker. Post 21 days of incubation, microbial biomass was removed from fungal isolates by passing the cultures through four layers of cheesecloth. The fatty acid concentration was minimized by the addition of 0.25 g sodium carbonate to culture filtrate and later extracted with two equal volumes of ethyl acetate. Under reduced pressure at 40 °C (vacuum evaporator), the solvent was removed, leaving behind dry solid residues which were re-dissolved in methanol. The crude extract containing taxol was subjected to TLC, HPLC, UV-spectroscopy, FTIR spectroscopy, MS, and NMR analysis for the presence, quantity, and purity of taxol by comparing with standard Paclitaxel procured from M.P Biomedicals (USA).
4
Preparation and Storage of Anticancer Agents
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The compound APR-246 and the proteasome inhibitor MG132 were purchased from MedChemExpress (China). Cycloheximide (CHX), carboplatin, doxorubicin, paclitaxel, and fluorouracil (5-FU) were purchased from MP Biomedicals (France). N-acetyl-L-cysteine (NAC) was bought from Beyotime Biotechnology (Beijing, China). For in vitro experiments, 100 mM APR-246 and 750 mM NAC were dissolved in DMSO, and ultrasonic was used for improving its solubility. MG132, CHX, and paclitaxel were dissolved in DMSO to 50 mg/mL, 50 mg/mL, and 100 mg/mL, respectively. Carboplatin (10 mg/mL), doxorubicin (100 mg/mL), and 5-FU (10 mg/mL) were dissolved in water. For in vivo experiments, APR-246 was rinsed with sterilized PBS and ultrasonized into clear solutions at concentration 100 mg/mL, and NAC was directly dissolved at 5 mg/mL in the drinking water. All the reagents were stored at -20°C for long-term preservation.
5
Comparative Cytotoxicity Assay for Kidney Cells
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Madin-Darby Canine Kidney cells (MDCK; CCL-34, ATCC, Manassas, VA, USA) were cultured at 37°C and 5% CO2 in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12; (Gibco, Life Technologies, Darmstadt, Germany) plus 5% fetal bovine serum (FBS; Gibco), 1% glutamine, and 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco). NRK-52E (CRL-1571, ATCC) cells were cultured in DMEM (Gibco) with 5% newborn calf serum (NBCS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C and 5% CO2. Human HK-2 cells (CRL-2190, ATCC) were cultured in DMEM with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO2. For the experiments, cells were first seeded into 6-well plates (Greiner Bio-One, Frickenhausen, Germany) for 24 h. Subsequently, cisplatin, PAC-1, doxorubicin (all from Tocris Bioscience, Bristol, UK), or paclitaxel (MP Biomedicals, Eschwege, Germany) were added for 24 h as indicated. For serum starvation, culture medium was replaced by serum free medium. After 24 h, cells were either trypsinated and counted with a Neubauer hemocytometer or analyzed for RNA isolation. Selective PPARγ inhibitor SR-202 (Biomol, Hamburg, Germany) was added to the culture medium along with cisplatin at 200 μM. Cell culture supernatants were collected and frozen for further use.
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