Human foreskin fibroblast (ATCC CRL 1475) cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, and 1% penicillin-streptomycin at 37°C in 5% CO2. The fibroblasts were plated in 24-well plates (Falcon™ Tissue Culture Plates) and Nunc™ 4-Well Dishes (Sigma-Aldrich) at a density of 2.5 × 105 per mL 24 hours prior to the start of the experiment to achieve 80-90% confluence. Eight hours prior to infection, half of wells in each plate were pre-treated/incubated with selected concentrations of metformin (0-50 ug/mL) (Sigma-Aldrich).
Tissue culture plates
Manufactured by Merck Group
Tissue culture plates are a type of laboratory equipment used for cell and tissue cultivation. They provide a sterile, controlled environment for the growth and maintenance of cells and tissues in vitro. These plates typically have a flat surface with individual wells or compartments, allowing for the culturing of multiple samples simultaneously. They are designed to support the attachment, proliferation, and differentiation of cells, making them an essential tool for various applications in cell biology, biomedical research, and pharmaceutical development.
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Lab products found in correlation
Metformin, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Nonessential amino acid, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Tylosine, by Merck Group (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Fibronectin coated, by Merck Group (1 mentions)
5 protocols using tissue culture plates
1
Fibroblast Culture and Metformin Treatment
2
Antifungal Susceptibility Profiling
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Minimal effective concentration (MEC) of caspofungin, a cell wall β-1,3-glucan synthesis inhibitor, and minimal inhibitory concentration (MIC) of posaconazole, SDS and H2O2 for the susceptibility to detergent and oxidative damages, were determined in MM-reza according to the Clinical Laboratory Standards Institute M38-A2 protocol (NCCM) by the microdilution method in 96-well plates [37 (link)].
To test the susceptibility to Congo red (CR) and calcofluor white (CFW), 500 conidia were spotted on six-well microplates (tissue culture plates, Sigma Aldrich) containing serial dilutions of CR or CWF in MM agar medium and incubated at 37 °C [36 (link)].
To test the susceptibility to Congo red (CR) and calcofluor white (CFW), 500 conidia were spotted on six-well microplates (tissue culture plates, Sigma Aldrich) containing serial dilutions of CR or CWF in MM agar medium and incubated at 37 °C [36 (link)].
3
Mouse and Human Cell Culture Protocols
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Mouse E14 embryonic stem cells (mESCs) and human embryonic kidney HEK293T cells were used for the experiments. mESCs were cultured on tissue culture plates (Sigma) pre-coated with 0.2% gelatin at 37°C in a 5% CO2 incubator. Cell culture media are consisted of 15% fetal bovine serum (FBS, Biowest), 1 mM sodium pyruvate (GIBCO), 100X non-essential amino acid (Corning), 0.1 mM β-mercaptoethanol (GIBCO), 100X GlutaMAX (GIBCO), Tylosine (Sigma), and 1,000 U/mL of LIF (conditioned media). LIF-containing media were produced from Cos7 cells transfected with LIF cDNA by Lipofectamine. Subculture was performed by 0.01% trypsin treatment for 3 min in a 37°C incubator. After incubation, trypsin was neutralized by FBS-containing media, and mESCs were collected by centrifugation. Subsequently, mESCs were plated on a pre-coated culture dish. HEK293T cells were maintained on a SPL tissue culture plate with Dulbecco’s Modified Eagle’s Medium (DMEM). Media contained 10% fetal bovine serum (FBS, Gibco) and 1X antibiotics (anti-anti, Gibco). Subculture was performed with 0.05% trypsin. Cells were re-suspended with FBS-containing media for neutralization and re-plated on a tissue culture plate. Calcium phosphate method was used for plasmid DNA transfection.
4
Neutrophil Migration Assay for Infants
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The assay was performed according to the principles outlined by Heit and Kubes (13 (link),15 (link)). Agarose gels were cast on tissue culture plates (Sigma-Aldrich) and stored overnight at 4 °C to make the gels firm and facilitate punching of the wells. Wells were punched the following day at a standardized distance of 2.5 mm from each other (Figure 1 ). Leukocytes from newborn infants and adults were added simultaneously to different tissue culture plates for each experiment. Leukocytes (1 × 105 per well) were added to each of the two outer wells (Figure 1 ) in order to demonstrate that the migration was directed towards the gradient of the central well. Chemoattractant or buffer was added to central wells. Gels were incubated for 2 hours at 37 °C, 5% CO2, whereby only neutrophils migrated outside the wells. Methanol (100%) was added to each well to terminate the migration and fix the cells. The gels were stored overnight at 4 °C.
5
Cardiomyocyte Isolation from Neonatal Mouse Hearts
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Cardiomyocytes were prepared from P0 1FF;2FF hearts of approximately 10 mouse pups. Ventricular tissues were predigested in trypsin (0.5 mg/ml; Sigma)–Hank’s balanced salt solution without Ca2+ at 4°C for 30 min with constant shaking. Cells were dissociated by four rounds of 4-min digestion with purified type II collagenase (Gibco) in Hank’s balanced salt solution at 37°C. Fibroblasts were removed from the cell suspension by two rounds of 1-hour differential plating at 37°C in Dulbecco’s modified Eagle medium/M199 (4:1 ratio) medium containing 10% horse serum, 5% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and Hepes (25 mM) in the presence of 5% CO2. Myocytes were cultured on fibronectin-coated (Sigma) tissue culture plates.
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