The ability of selected drugs to affect mammosphere-forming efficiency (MSF) and mammosphere viability (MSV) was studied in ultra-low attachment 96-well plates (Corning®, One Riverfront Plaza Corning, Corning, NY, USA). Cells were plated at low density (1000 viable cells/well) in serum-free RPMI 1640 media supplemented with 2% antibiotic-antimycotic mixture and additional factors enhancing CSC maintenance [12 (link),20 (link)]. In detail, glucose (60 mg/mL), L-glutamine (10 µL/mL), heparin (4 µg/mL), BSA (2 mg/mL), EGF (0.02 µg/mL), FGFb (0.01 µg/mL), putrescin (10 µg/mL), apo-transferrin (0.1 mg/mL), insulin (25 µg/mL), 30 µM selenium and 20 µM progesterone (all from Sigma-Aldrich, St. Louis, MO, USA). Twenty-four hours after seeding, the cells were treated with drugs for 7 days. The formed spheres were observed by the optical microscope (Olympus IMT-2 model) and quantified by presto blue (Invitrogen, Thermo Fisher Scientific, Madrid, Spain) to further calculate IC50 as previously described [12 (link),25 (link)].
Putrescin
Manufactured by Merck Group
Sourced in France
Putrescin is a laboratory instrument designed for the detection and quantification of the chemical compound putrescine. Putrescine is a diamine compound that is produced during the decomposition of organic matter, particularly in animal tissues. The Putrescin instrument provides a reliable and accurate method for the analysis of putrescine levels in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (4 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Selenium, by Merck Group (2 mentions)
Progesterone, by Merck Group (2 mentions)
Apo transferrin, by Merck Group (2 mentions)
Donor calf serum, by Thermo Fisher Scientific (2 mentions)
Heparin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Prestoblue, by Thermo Fisher Scientific (1 mentions)
Ultra low attachment 96 well plate, by Corning (1 mentions)
Laminin, by Merck Group (1 mentions)
Igf 1, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Thermo Fisher Scientific (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
μ dish, by Ibidi (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Dispase, by STEMCELL (1 mentions)
Primocin, by InvivoGen (1 mentions)
7 protocols using putrescin
1
Mammosphere Formation Assay Protocol
2
Isolation and Culture of Rat Tanycytes
3
Neural Differentiation of hiPSCs
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Neural differentiation followed previously published protocols (Hu and Zhang, 2009 (link); Stockmann et al., 2013 (link)). hiPSCs were lifted using dispase (Stemcell Technologies) and grown as EBs in low-attachment flasks (Corning Costar) using hESC medium. ROCK inhibitor (Ascent Scientific) was added the first 24 h. Medium was changed to neuronal basal-medium [DMEM/F12, 0.02 mg/ml insulin (SAFC), 24 nM sodium selenite, 16 nM progesterone, 0.08 mg/ml apotransferrin, 7.72 μg/ml putrescin, 50 mg/ml heparin (all Sigma-Aldrich), 1% NEAA, 1% antibiotic-antimycotic, 10 μg/ml BDNF, GDNF, and IGF-1 (all Gibco), 0.1 μMcAMP (Sigma-Aldrich), 50 mg/ml ascorbic acid (PeproTech)] at Day 3 of culture. At Day 7, EBs were plated on laminin (Sigma)-coated dishes. 0.1 μM retinoic acid was added from Day10 and B27 (Gibco) and 1 μM purmorphamine (Calbiochem) from Day 14, when neural rosettes were detached and grown in suspension again. At Day 28, EBs were plated on 35 mm μ-dish (Ibidi) coated with poly-L-ornithine (Sigma) and laminin. The medium was changed to 0.5 μM purmorphamine and 0.05 μM retinoic acid and changed one time per week. Neuronal cells were harvested or fixated at Days 21 and 42 after final plating.
4
Isolation and Culture of Rat Tanycytes
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Tanycytes were isolated from the median eminence of the hypothalamus of 10-d-old rats and cultured as described previously 54 (link),84 (link). Briefly, after decapitation and removal of the brain, median eminences were dissected and crushed on 80μM nylon mesh (Sefar America Inc., Kansas City, MO). Dissociated cells were cultured in DMEM/F12 (Invitrogen, Cergy Pontoise, France) supplemented with 10% (v/v) donor calf serum (Invitrogen) under humid atmosphere of 5 % CO2-95 % air at 37 °C. Culture medium was changed after 3-4 days of culture and subsequently every 2 days. Upon reaching confluence, the tanycytes were isolated from contaminating cells by overnight shaking at 250 rpm at 37 °C and either replated in 6 cm dishes for Western blot experiments or seeded in culture plates on poly-L-lysine-coated glass coverslips for studying leptin traficking. Two days before treatment, the medium was replaced by a tanycyte defined medium (TDM) consisting of DMEM/F12 (devoid of phenol red; Invitrogen) supplemented with insulin (5μg/ml) (Sigma, Saint Quentin Fallavier, France) and putrescin (100μM) (Sigma).
5
Establishing Zebrafish Patient-Derived Xenografts
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Human samples used for zebrafish patient-derived xenograft (zAvatars) establishment were obtained from Champalimaud Hospital and Prof Fernando Fonseca Hospital with written informed consent. The study was approved by both Hospital Ethics Committees.
Neoplastic colorectal tissues were obtained from surgically resected specimens. Human tissue processing protocol was performed as previously described15 (link). In brief, samples were washed in ice-cold 1X-PBS, chopped into small pieces, and cryopreserved in 90% FBS 10% DMSO. Cryopreserved human primary tumor tissue was defrosted, further washed, and minced in mix1 (DMEM-F12 (Gibco), 60%FBS (Sigma), Y-27632 10 μM (Cliniscience), Primocin 100 μg/ml (Invivogen), Putrescin 10 μg/ml (Sigma-Aldrich), Nicotinamide 10 mM (Sigma-Aldrich), and digested with Liberase (Roche) for 5–10 min at 37 °C. Tumor cell suspension was passed through a 70 μm cell strainer and centrifuged at 250 × g for 4 min at 4 °C. For cell labeling, tumor cells were incubated with CM-DiI (1:100) in mix1 but without FBS and supplemented with DNase I 5 U/ml (Fermentas), for 15 min at 37 °C. Cells were resuspended in mix1 supplemented with human EGF (50 ng·mL−1, Peprotech) at final concentration of ~0.25 × 106 cells per milliliter.
Neoplastic colorectal tissues were obtained from surgically resected specimens. Human tissue processing protocol was performed as previously described15 (link). In brief, samples were washed in ice-cold 1X-PBS, chopped into small pieces, and cryopreserved in 90% FBS 10% DMSO. Cryopreserved human primary tumor tissue was defrosted, further washed, and minced in mix1 (DMEM-F12 (Gibco), 60%FBS (Sigma), Y-27632 10 μM (Cliniscience), Primocin 100 μg/ml (Invivogen), Putrescin 10 μg/ml (Sigma-Aldrich), Nicotinamide 10 mM (Sigma-Aldrich), and digested with Liberase (Roche) for 5–10 min at 37 °C. Tumor cell suspension was passed through a 70 μm cell strainer and centrifuged at 250 × g for 4 min at 4 °C. For cell labeling, tumor cells were incubated with CM-DiI (1:100) in mix1 but without FBS and supplemented with DNase I 5 U/ml (Fermentas), for 15 min at 37 °C. Cells were resuspended in mix1 supplemented with human EGF (50 ng·mL−1, Peprotech) at final concentration of ~0.25 × 106 cells per milliliter.
6
Purification and Assay of Vegetal Diamine Oxidase
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Vegetal diamine oxidase from Pisum sativum (Diamaze 1.2 U/mg protein) was from IBEX Pharmaceuticals Inc. (Montreal, QC, Canada) and kept at −80 °C. Monobasic and dibasic phosphate, 4-Aminoantipyrine (AAP), Sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DCHBS), horseradish peroxidase (HRP), Putrescin, Ammonia assay kit, Bovine Serum Albumin (BSA), sucrose, trehalose, carboxymethyl cellulose (CMC), Mg stearate, pepsin from porcine mucosa (460 units/mg solid), and pancreatin from porcine pancreas (8X USP specifications) were purchased from Sigma Aldrich (Oakville, ON, Canada). The Bradford reagent was purchased from BioShop® (Burlington, ON, Canada). The Hydroxypropyl Methyl Cellulose (HPMC) K100 was gifted by Colorcon (Harleysville, PA, USA).
7
Enrichment of Breast Cancer Stem Cells
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MDA-MB-231 breast cancer cell line was routenely maintained in RPMI 1640 supplemented with 10 % fetal bovine serum (FBS) and 6 mM GlutaMAX (Invitrogen). Breast cancer cell lines naturally contain CSC. 21 (link) To facilitate the enrichment of CSC within the MDA-MB-231 cell line, mammosphere were cultured as previously described 22 (link) in low attachment plates (Corning) in serum-free RPMI 1640 supplemented with 60 mg/mL glucose, 10 µL/mL L-glutamine, 10 µL/mL antibiotic-antimitotic mixture (Life Technologies), 0.01 μg/mL FGFb (Thermo Fisher Scientific), and 4 μg/mL heparin, 2 mg/mL BSA, 0.02 µg/mL EGF, 10 μg/mL putrescin, 0.1 mg/mL apo-transferrin, 25 μg/mL insulin, 30 μM selenium, and 20 μM progesterone (all from Sigma).
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