The RH30, GFP+ RH30, and RD spheroids were obtained seeding 5 × 103 cells in ultralow-adhesion, round-bottom 96-well plates (Corning, Tewksbury, USA) in low glucose DMEM supplemented with B27 (both from Gibco, Monza, Italy), 10 ng/ml bFGF and 20 ng/ml EGF (both from ORF Genetics, Kopavogur, Iceland). HT29 spheroids were produced seeding 5 × 103 cells in the same plate in high glucose DMEM (Gibco, Monza, Italy) supplemented with 5% FBS (Gibco, Monza, Italy). After 5 days, cell viability was assessed using Cell Titer-Glo Luminescent assay kit (Promega, Madison, USA). Five days after seeding, each spheroid was transferred on a layer of 1 × 104 BJ or MSC. These cells were used as stromal layer and the spheroid-stromal interaction was monitored for 48 h using Leica DMI6000B microscope. Cytotoxicity was analyzed with the Lactate Dehydrogenase Activity (LDH) Assay Kit (Sigma-Aldrich, Saint Louis, USA). Supernatant of stromal cells (BJ and MSC) and of spheroids alone was used as control. Absorbance was expressed as fold change in respect to the control. Alive cells were detected with Live and Dead staining (Thermo Fisher, Waltham, USA) following manufacturer’s instructions. Five experiments were performed for each condition.
Lactate dehydrogenase ldh activity assay kit
Manufactured by Merck Group
Sourced in United States
The Lactate dehydrogenase (LDH) activity assay kit is a laboratory tool used to measure the activity of the LDH enzyme. LDH is an important enzyme involved in energy metabolism and is commonly used as a biomarker in various clinical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Celltiter glo luminescent assay kit, by Promega (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Live and dead staining, by Thermo Fisher Scientific (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Trypan blue exclusion assay kit, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Deuterium oxide d2o, by Cambridge Isotopes (1 mentions)
Ham s f 12k medium, by Merck Group (1 mentions)
Potassium phosphate monobasic and dibasic, by Merck Group (1 mentions)
N methyl n trimethylsilyl trifluroacetamide, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Tetrahydrofuran, by Merck Group (1 mentions)
A549 human pulmonary epithelial cell line, by American Type Culture Collection (1 mentions)
10 protocols using lactate dehydrogenase ldh activity assay kit
1
Spheroid-Stromal Interaction Assay
2
Evaluating Essential Oil Compounds on MC3T3-E1 Cell Growth
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MC3T3-E1 cells were seeded in 96-multiwell plates in 100 μL of complete culture medium at a concentration of 0.5 × 105 cells/mL. After 24 h incubation at 37 °C, the culture medium was replaced with 100 μL of the highest non-toxic concentrations of each essential oil-derived compound. After 24 h and 72 h exposure of cells to the compounds, MC3T3-E1 cells were lysed, and the exact cell number was calculated based on the release of total LDH from the cell population using the Lactate Dehydrogenase Activity (LDH) Assay Kit (Sigma-Aldrich Chemicals, Warsaw, Poland). In this test, unlike a typical LDH cytotoxicity assay, the higher the content of released LDH observed, the greater number of cells present in the population. The experiment was performed according to the manufacturer’s protocol. The cell concentration after 24 h and 72 h cultures was estimated from the calibration curve made for the known number of lysed MC3T3-E1 cells. The doubling time (DT) for the cells was calculated using Doubling Time Computing software version 3.1.0.
3
Evaluating Enoxaparin and UFH Effects on A549 Cells
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A549 human pulmonary epithelial cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Enoxaparin was obtained from Aventis Pharma Ltd. (NSW, Australia). UFH was purchased from Hospira Pty. Ltd. (Victoria, Australia). Sodium chloride, tetrahydrofuran, sodium hydroxide, N-methyl-N-(trimethylsilyl) trifluroacetamide, methanol, acetic acid, Ham’s F12K medium, antibiotics (penicillin G and streptomycin), trypsin-ethylenediaminetetraacetic acid (EDTA), trypsin, thrombin, enzyme-linked immunosorbent assay (ELISA) kits for IL-6 and IL-8, trypan blue exclusion assay kit, lactate dehydrogenase (LDH) activity assay kit, and potassium phosphate monobasic and dibasic were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Fetal bovine serum, IL-6 and IL-8 recombinant human proteins were obtained from Invitrogen (Grand Island, NY, USA). Deuterium oxide (D2O) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA).
4
Antioxidant Assays and Plasmid DNA Protection
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2, 4, 6-Tri (2-pyridyl)-s-triazine (TPTZ) were purchased from Fluka (Switzerland). 2,2′-azino-bis (3-ethylbenothiazoline-6-sulfonic acid) diammonium salts (ABTS), 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), the homologous series of n-hexane (C8-C24) and lactate dehydrogenase (LDH) activity assay kit were from Sigma (USA). Propidium iodide (PI) was from BD Biosciences. The pBR322 plasmid DNA was from Takara Bio Co. Ltd. (Dalian, China). Other chemicals used were all of analytical grade and obtained from China.
5
Evaluating Metabolic Enzyme Activity
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For analysis of metabolic enzyme activity, 5 × 104 cells were cultured and treated as indicated, then subjected to analyze activities of metabolic enzymes with specific assay kits, according to the manufacturer’s instructions. All used kits were listed as follows: Hexokinase (HK) Activity Assay Kit (Sigma, MAK091-1KT), Phosphoglucose Isomerase (GPI/PGI) Activity Assay Kit (Sigma, MAK103-1KT), Phosphofructokinase (PFK) Activity Assay Kit (Sigma, MAK093-1KT), Fructose-bisphosphate Aldolase (ALDO) Activity Assay Kit (Sigma, MAK223-1KT), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Activity Assay Kit (BioVision, K680-100), Enolase (ENO) Activity Assay Kit (Sigma, MAK178-1KT), Pyruvate Kinase (PK) Activity Assay Kit (Sigma, MAK072-1KT), Pyruvate Dehydrogenase (PDH) Activity Assay Kit (Sigma, MAK183-1KT), Lactate Dehydrogenase (LDH) Activity Assay Kit (Sigma, MAK066-1KT), Glutaminase (GLS) Activity Assay Kit (Nanjing Jiancheng Bioengineering Institute, A124) and Glutamine Synthetase (GLUL/GS) Activity Assay Kit (Nanjing Jiancheng Bioengineering Institute, A047). Enzyme activity was determined using an iMarkTM Microplate Reader (Bio-Rad, 168–1130). The values were normalized to the protein concentration.
6
PET Film Characterization Protocol
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PET films of 100-micron thickness were kindly gifted by Sumilon Polyester Ltd. (India). Cysteine, 5,5′-Dithiobis (2-nitrobenzoic acid) (Ellman’s reagent), Griess reagent (modified) kit, lactate dehydrogenase (LDH) activity assay kit and bicinchoninic acid (BCA) kit were purchased from Sigma-Aldrich. Analytical grade reagents were used for all other experiments unless otherwise specified.
7
Cell Proliferation and Cytotoxicity Assays
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Cell Proliferation Kit I (MTT) and Lactate Dehydrogenase (LDH) Activity Assay Kit were from Sigma-Aldrich (St. Louis, MO, USA).
8
Cytotoxicity Evaluation of Chemical Agents
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MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] powder was provided by Sigma Chemical Co. (St. Louis, MO, USA). Lactate dehydrogenase (LDH) activity assay kit (Catalog Number MAK066) was purchased from Sigma Aldrich (Sigma Aldrich, USA). Primary and secondary antibodies were supplied by Santa Cruz Biotechnology and Abcam companies (USA, England). NaOCl and curcumin were provided by Nik Darman Asia and Safir Azma companies (Iran), respectively. All cell culture materials were supplied by appropriate, commercially available suppliers.
9
Histological Evaluation of Lung Pathology
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For analysis of lung pathology, lung tissues were fixed in 10% formalin and 5 μm sections were prepared and stained with hematoxylin and eosin (H&E). A scoring system was used based upon previous influenza studies [44 (link)–46 (link)]. Ten fields of lung sections were randomly chosen and scored as previously described [47 (link)]. Images were captured using an Olympus BX41 microscope under 20X magnification and CellSense software. Total protein concentrations in BALF were analyzed using a Pierce BCA protein assay kit (Thermo Scientific). Nitrite levels were quantified using a Griess kit (Life Technologies). The BCG albumin assay kit (Sigma-Aldrich) was used to determine albumin and the Lactate Dehydrogenase (LDH) Activity Assay Kit (Sigma-Aldrich) was used to assess LDH levels.
10
Biomaterial Synthesis and Characterization
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Poly(glycerol sebacate) (PGS) prepolymer was synthesized from sebacic acid (Sigma-Aldrich) and glycerol (Fisher Scientific) using a published protocol [38] . Type I collagen was purchased commercially (Elastin Products Company, Inc). Silk fibroin was extracted from raw silk (Haian Silk Company, Nantong, China) according to published methods [39] . Recombinant human syndecan-4 was obtained commercially (R&D Systems). The human complement C3 ELISA kit and the human complement C3a des Arg ELISA kit were purchased from Abcam. The lactate dehydrogenase (LDH) activity assay kit was purchased from Sigma-Aldrich. ePTFE vascular grafts were obtained from GORE-TEX® (Newark, Delaware). Bovine pericardial patch for valve replacement (BPV) was obtained from Edwards Lifesciences Corp. (Irvine, CA).
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