Primary MLFs were derived from Ahr+/+ and Ahr−/− mice as previously described21 (link). All animal procedures were approved by the McGill University Animal Care Committee (Protocol Number: 5933) and were carried out in accordance with the Canadian Council on Animal Care and ARRIVE guidelines22 (link). Fibroblasts were cultured in Gibco MEM (Thermo Fisher Scientific) supplemented with 10% FBS (Wisent Bioproducts), 1X Gibco GlutaMAX (Thermo Fisher Scientific), 1X antibiotic–antimycotic solution (Wisent Bioproducts: #450115), and 0.05 mg/ml gentamycin sulfate solution (Wisent Bioproducts). Cells were maintained at 37 °C in humidified air with 5% CO2. Cells used in each experiment were within two passage difference and no fibroblasts exceeding passage ten were used. All cells were seeded at 10,000 cells/cm2, grown to approximately 80–90% confluence and cultured in serum-free medium for 18 h before conducting the experiments unless otherwise indicated.
Gibco mem
Manufactured by Thermo Fisher Scientific
Sourced in United States
Gibco MEM is a basal medium used to support the growth and maintenance of a variety of cell types in cell culture. It provides essential nutrients and a balanced salt solution to support cell viability and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Gibco glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
Antibiotic antimycotic solution, by Wisent (1 mentions)
L glutamine, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Merck Group (1 mentions)
Centrifuge 5810r, by Eppendorf (1 mentions)
24 well plate, by BD (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
8 protocols using gibco mem
1
Fibroblast Culture from Ahr Mice
2
Cell Culture and Toxin Treatment
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C6 glioma cells (a gift from Prof. Colin Taylor, University of Cambridge) were cultured in Gibco® MEM (Thermo Fisher, UK) supplemented with 10% foetal bovine serum (FBS, Sigma, UK), 2 mM L-glutamine (Sigma, UK), and 1% antibiotic/antimycotic (Sigma, UK). ST14A cells (rat-derived striatal cells (Tissue and Cell Biotechnologies, Italy) were grown in Gibco® DMEM/F12 1:1 (1X) -Glutamax TM (Thermo Fisher, UK), supplemented with 10% FBS and 1% antibiotic/antimycotic. C6 cells were maintained at 37 C in humidified 95% air and 5% CO2. ST14A cells were grown at 33 C in humidified 95% air and 5% CO2 because propagation of ST14A cells at 37 C has been shown to induce differentiation into glial cells (Ehrlich et al. 2001) . Where appropriate cells were treated with pertussis toxin (PTX, Thermo Fisher, UK) at a range concentration of 2 pg/ml to 200 ng/ml or cholera toxin (CTX, Sigma, UK), at a concentration of 3.5 pg/ml to 350 ng/ml. PTX uncouples receptor-mediated Gαi-dependent inhibition of cAMP production (Weston et al. 2016) . CTX inhibits GTPase activity of Gs and causes permanent activation.
3
Quantifying Cytomegalovirus Infection in Fibroblasts
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Human foreskin fibroblasts were seeded into 24 well plates (Falcon, BD, New Jersey, NJ) and were maintained in minimal essential medium until further use (Gibco MEM supplemented with 5% fetal calf serum, Thermo Fischer). The medium was removed before inoculation and 100 μL of processed HM specimen was added to each well. Inoculated plates were centrifuged at 4°C and 900 g for 50 min (Centrifuge 5810 R, Eppendorf, Hamburg, Germany).
After centrifugation virus inoculum was removed, 1 mL of minimal essential medium was added to each well and the cultures were incubated at 37°C and 5% carbon dioxide for 18 h. Afterwards, monolayers were washed twice in phosphate-buffered saline and fixed with ice-cold acetone for 20 min. Cell monolayers were then incubated with an anti-CMV immediate early antigen (IEA) monoclonal antibody (bioMérieux, Lyon, France) and, after washing, with a fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G conjugate (Agilent, Santa Clara, CA). Read-out was the number of CMV specific IEA stained nuclei/mL whey. Duplicates for each sample were analyzed.
After centrifugation virus inoculum was removed, 1 mL of minimal essential medium was added to each well and the cultures were incubated at 37°C and 5% carbon dioxide for 18 h. Afterwards, monolayers were washed twice in phosphate-buffered saline and fixed with ice-cold acetone for 20 min. Cell monolayers were then incubated with an anti-CMV immediate early antigen (IEA) monoclonal antibody (bioMérieux, Lyon, France) and, after washing, with a fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G conjugate (Agilent, Santa Clara, CA). Read-out was the number of CMV specific IEA stained nuclei/mL whey. Duplicates for each sample were analyzed.
4
Mammalian Ear Pinnae DNA Extraction
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Ear pinnae from small mammals were homogenized with two stainless steel beads and 1 ml of cell culture medium (Gibco™ MEM, ThermoFisher Scientific, Massachusetts, United States) using the TissueLyser II (2 min at 30 Hz) (Qiagen, Hilden, Germany). The homogenized samples were centrifuged for 5 min at 20,000×g.
DNA was isolated from 350 µl of the supernatant using QiAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored in aliquots at − 20 °C.
DNA was isolated from 350 µl of the supernatant using QiAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored in aliquots at − 20 °C.
5
Isolation and Culture of Rat Hippocampal Neurons
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Rat primary hippocampal neurons were obtained from neonatal (P0–P1) Wistar rats. Animals were decapitated, and both of the hippocampi were dissected out and dissociated using papain latex. Then, neuronal cells were plated at a density of 2 × 105 cells/mL in Gibco™ MEM supplemented with 10% bovine calf serum, 1% Gibco™ MEM Non-Essential Amino Acids, 1% GlutaMAX, 1% sodium pyruvate, 0.3% glucose, 100 units/mL penicillin, and 0.1 mg/mL streptomycin and were maintained in a humidified incubator with 95% O2/5% CO2 at 37°C on 3.5 cm2/well plates that were previously coated with 50 μg poly-d -lysine/well. Then, 2–3 h after plating, MEM was replaced with Neurobasal Medium that supplemented with 2% B27 supplement, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 1% glutamine, 0.5% glutamic acid, and 25 μM β-mercaptoethanol. On the third day after seeding, 5 mM cytosine-β-d-arabinofuranoside was added to the cultures to inhibit the proliferation of non-neuronal cells. To maintain the cultures, it was necessary to replace the medium containing supplements every 3–4 days.
6
Cell Viability in Acoustically-Exposed Hydrogels
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Cell viability was assessed by performing a live/dead stain after culture of cells in the cross-linked hydrogel droplets at 37 ºC in 5% CO2. Cell viability was measured after 1 day, 3 days and 7 days of culture for cells exposed to the acoustics. As a control group cell viability was measured after 7 days of culture for cells not exposed to the acoustics. The hydrogel droplets were washed twice with MEM transparent media (Gibco MEM, no glutamine, no phenol red, Fisher Scientific) by gently pipetting up and down and centrifuging at 500 rpm. Staining was performed by incubating the cell-laden hydrogel droplets for 15 min with 2 µM calcein AM (Fisher Scientific) and 1 µM propidium iodide (Fisher Scientific) in MEM. Cells were washed with MEM once and visualised immediately after staining. Cell viability was measured by manually counting the number of living and dead cells in the droplets. For each experimental group 197–214 cells were counted.
7
MDCK Cell Culture Protocol
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Madin‐Darby Canine Kidney (MDCK) cells were cultured in MEM (GIBCO, Life Technologies) with 10% FBS, 1% L‐glutamine and antibiotics.
8
Gnotobiotic Pig Intestinal Viral Isolation
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Previously collected gnotobiotic pig small intestinal contents containing RVA G9P[13] (G9P[13] strain) and RVA G5P[7] were used in the study. Intestinal contents were diluted at a 1:10 ratio in sterile minimal essential media (MEM Gibco; Life Technologies, Grand Island, NY, USA). Contents were then centrifuged at 2095× g for 10 min at 4 °C and the supernatants filtered through a 0.2 µm filter.
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