Alveolar epithelial cells were isolated from the lungs of Sox2SPC-rtTA mice by Dispase (BD, Pharmingen) digestion as described previously with few modifications [43] (link). Lungs were exsanguinated by perfusing through the right ventricle with 4 ml PBS after opening the peritoneum, clipping the vena cava inferior and removing the ribcage. 1 ml Dispase (BD, Pharmingen) was instilled over a tracheal cannula into the lung, immediately a sterilized suture (Braun) was used to tighten a node around the cannulised trachea. Lungs were isolated, incubated for 45 minutes in 1 ml Dispase at room temperature and transferred to a culture dish containing 5 ml DMEM/F12 medium (Gibco) supplemented with 0.04 mg/ml DNase I (AppliChem), 3.6 mg/ml D-(+)-Glucose (AppliChem) and 1% Penicillin/Streptomycin (P/S). The small airways were gently removed and the obtained cell suspension was serially filtered through 100, 70 and 40 µm nylon meshes and centrifuged at 200 g for 10 minutes at 15°C. The supernatant was discarded and the cell pellet was resuspended in 500 µl DMEM/F12 (Gibco) medium supplemented with 3.6 mg/ml D-(+)-Glucose (AppliChem), 1% P/S and 2% FCS. AVTII cells were cultured in DMEM/F10 containing 10% FCS and 1% P/S in tissue culture cover slip immersed in 12 well plates (Corning, NY) previously coated with collagen (Inamed); cultures were maintained in a 5% CO2/air incubator.
D glucose
Manufactured by AppliChem
Sourced in Germany
D-glucose is a simple sugar molecule that serves as a fundamental building block for various carbohydrates. It is the primary source of energy for many living organisms and plays a crucial role in cellular metabolism.
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Lab products found in correlation
Penicillin streptomycin solution, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Hepes, by AppliChem (2 mentions)
Celltiter 96 aqueous mts, by Promega (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Sodium pyruvate, by Lonza (2 mentions)
Absolute ethanol, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sodium bicarbonate, by Merck Group (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Formalin, by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Alexa fluor 647 conjugated anti mouse cd62p, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin 0.25, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (1 mentions)
Kynuric acid, by Merck Group (1 mentions)
Cacl2, by Carl Roth (1 mentions)
6 protocols using d glucose
1
Isolation of Alveolar Epithelial Cells from Mouse Lungs
2
Platelet Count and Activation Assay
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The platelet count and activation state were determined using flow cytometry following an optimized protocol based on Alugupalli et al. (2001) (link). In brief, 5 μL of tail blood was collected into 45 μL 0.11 M sodium citrate (Merck) in 0.01 M PBS, pH 6.5. Diluted whole blood (10 μL) was stained with 5 μL each of anti-mouse CD61 PE (Thermo Fisher Scientific) and Alexa Fluor 647-conjugated anti-mouse CD62P (BD Pharmingen) for 30 min. The samples were fixed with 975 μL platelet fixation solution (0.01 M PBS containing 0.2% BSA [Sigma-Aldrich], 0.1% D-glucose [AppliChem], and 0.1% formalin [Sigma-Aldrich]). A dose of 5 μL of Sphero 8-peak Rainbow Calibration Particles (3.0–3.4 μm diameter; BD Biosciences) was added as an internal standard to determine the platelet count. A total of 30,000 platelet events were analyzed by flow cytometry. Activated platelets were identified as CD62P+ cells within the CD61+ population.
3
Cytotoxicity Assay Using Cell Lines
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Cell culture media (McCoy’s 5A and DMEM), FBS (fetal bovine serum), trypsin (0.25%), penicillin-streptomycin solution, and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich (Barcelona, Spain). D-glucose was acquired from AppliChem (Barcelona, Spain). CellTiter 96®AQueous Non-Radioactive Cell Proliferation Assay (used for the cellular viability assays) was purchased from Promega (Lisbon, Portugal).
4
Organotypic Slice Culture Preparation
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OSCs were prepared as described by [45 (link)]. In brief, brains from 2 to 3 month old C57BL/6 mice were removed and immediately immersed in ice-cold Ringer solution [2.5 mM KCl (Merck KGaA, Darmstadt, Germany), 1 mM MgCl2 (Sigma-Aldrich), 260 mM d -Glucose (Applichem), 26 mM NaHCO3 (Merck KGaA), 1.25 mM NaH2PO4 (Merck KGaA), 2 mM pyruvic acid (PAA Laboratories), 3 mM myo-inositol (Sigma-Aldrich), 1 mM kynuric acid (Sigma-Aldrich), 2 mM CaCl2 (Carl Roth GmbH), pH 7.3]. The brains were embedded in 4% low melting agarose (Sigma-Aldrich). After polymerization agarose blocks were trimmed and glued onto the cutting table of a vibratome (VT1000, Leica, Heerbrugg, Switzerland). Brains were cut in coronal slices of 250 µm. Slices were then collected and stored in ice-cold Ringer solution before floating onto semi-porous membrane inserts (Millipore, 0.4 mm pore diameter) according to Stoppini et al. [46 (link)]. The slices were cultivated in wells of a 6-well plate at 37°C under 5% CO2 in a standard medium consisting of DMEM/Ham’s F12 (pH 7.3; PAA Laboratories), 24% normal horse serum (PAA Laboratories), 2% HEPES (PAA Laboratories), 0.1% gentamycin (PAA Laboratories) and additional 10 mM d -glucose (Sigma-Aldrich). Medium was changed every other day and viability of brain slices was assessed using PI staining.
5
Cytotoxicity Evaluation of MnTnHex and Cisplatin
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RPMI-1640 with L-glutamine was purchased from Biowest (Nuaillé, France). MnTnHex was synthesized and characterized at Duke University School of Medicine, according to Batinic-Haberle et al. [35 (link)]. Cisplatin, penicillin-streptomycin solution (10,000 units/mL of penicillin; 10 mg/mL of streptomycin), crystal violet, sodium bicarbonate, and O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA, ≥99%) were obtained from Sigma-Aldrich (Madrid, Spain). Ethanol absolute and acetic acid were purchased from Merck (Darmstadt, Germany). Sodium pyruvate was purchased from Lonza (Basel, Switzerland) and trypsin (0.25%), and fetal bovine serum (FBS) from Gibco (Eugene, OR, USA). HEPES and D-Glucose were obtained from AppliChem (Darmstadt, Germany). CellTiter 96® Aqueous MTS (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was acquired from Promega (Madison, WI, USA).
The stock solution of MnTnHex and its dilutions were prepared in Milli-Q water. Cisplatin was dissolved in saline solution (0.9% NaCl), and its aliquoted solutions were stored at −20 °C. In all cell-based assays, controls were also included, in which cells were exposed to either saline solution or Milli-Q water.
The stock solution of MnTnHex and its dilutions were prepared in Milli-Q water. Cisplatin was dissolved in saline solution (0.9% NaCl), and its aliquoted solutions were stored at −20 °C. In all cell-based assays, controls were also included, in which cells were exposed to either saline solution or Milli-Q water.
6
Cytotoxicity Evaluation of MnBuOE
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D-Glucose and HEPES were acquired from AppliChem (Darmstadt, Germany). Cisplatin, crystal violet (CV), extracellular matrix (ECM), penicillin–streptomycin (Pen/Strep) solution (10,000 units/mL of penicillin; 10 mg/mL of streptomycin) and sodium bicarbonate were obtained from Merck (Madrid, Spain). RPMI-1640 medium with L-glutamine was purchased from Biowest (Nuaillé, France). FBS and Trypsin (0.25%) were obtained from Gibco (Eugene, OR, USA). Sodium pyruvate was acquired from Lonza (Basel, Switzerland). CellTiter 96® Aqueous MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-Tetrazolium) was obtained from Promega (Madison, WI, USA). Acetic acid and ethanol absolute were acquired from Merck (Darmstadt, Germany).
MnBuOE was synthesized and characterized at Duke University School of Medicine. Stock solutions and respective dilutions were formulated in Mili-Q water. Cisplatin was dissolved in saline solution (0.9% NaCl) and its aliquoted solutions were kept at −20 °C. In all cell-based assays, controls were also included, in which cells were exposed to saline solution or Milli-Q water.
MnBuOE was synthesized and characterized at Duke University School of Medicine. Stock solutions and respective dilutions were formulated in Mili-Q water. Cisplatin was dissolved in saline solution (0.9% NaCl) and its aliquoted solutions were kept at −20 °C. In all cell-based assays, controls were also included, in which cells were exposed to saline solution or Milli-Q water.
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