Scratch assays were performed at passage 2 in a 6‐well cell culture plate (Greiner Bio‐One GmbH, Frickenhausen, Germany) to which a sterile culture insert (ibidi GmbH, Martinsried, Germany) was added. A 500‐μm thick wall separated each culture into two 70 μL cell culture reservoirs. MSCs (supplemented with FCS, SHS or AS) were plated at a density of 30 000 cells/70 μL well and cells were cultivated for 24 h at 37°C and 5% CO2. Subsequently, the culture insert was removed creating a cell‐free gap. Cells were further cultivated in a life cell imaging system (Cell Observer® Systems, Zeiss MicroImaging). Cell‐migration and cell‐proliferation was continuously recorded for 24 h with the computer program Axio Vision. Finally, the gap area overgrown by cells was determined using the picture processing software Photoshop (Adobe Photoshop, CS3; Fig. 5 ).
6 well cell culture plate
Manufactured by Greiner
Sourced in Germany
The 6-well cell culture plates are a laboratory equipment used for the in vitro cultivation of cells. Each plate contains six individual wells, providing a controlled and sterile environment for cell growth and experimentation.
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Lab products found in correlation
Dm il led microscope, by Leica (1 mentions)
Sa β gal assay kit, by Cell Signaling Technology (1 mentions)
Matrigel matrix, by Corning (1 mentions)
Mtesr1, by STEMCELL (1 mentions)
Dispase, by STEMCELL (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Essential amino acid, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Dmem glutamax growth medium, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Versene solution, by Thermo Fisher Scientific (1 mentions)
Essential 8 medium e8, by Thermo Fisher Scientific (1 mentions)
Matrigel hesc qualified matrix, by Corning (1 mentions)
175 cm2 flasks, by Greiner (1 mentions)
Gentamicin, by Merck Group (1 mentions)
7 protocols using 6 well cell culture plate
1
Scratch Assay for MSC Migration and Proliferation
2
Optimized Blue Light Irradiation Protocol
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As blue light emitting source we used a 12 x 10 cm LED-arrays with 60 LEDs emitting quasimonochromatic light with a maximum intensity at 453 ± 10 nm (royal blue). The light sources used here were designed by Philips Research (Aachen, Germany) and the irradiance of the LED arrays was characterized using an Ulbricht sphere. Cell cultures in the current study were irradiated with an irradiance of 39 mW / cm2 at a distance of 5 cm. Routinely, we tested the heat development during the irradiation of 2 ml PBS in a 6-Well cell culture plate (Greiner Bio-One GmbH; Kremsmünster, Austria) corresponding to our experimental setup. We observed an increase in temperature of 1-2°C during the 10 minutes light exposure but sample temperature never exceeded 33°C. The degree of evaporation we determined as the result of the irradiation was so low that the possible osmotic effects of the light-exposed sample were negligible. In order to achieve comparable conditions, cell culture plates containing the control samples were located in a windowed heating cabinet at 33°C.
3
Quantifying Cellular Senescence with SA-β-gal
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For the SA-β-gal assay, transfected cells were seeded in 6-well cell culture plates (Greiner) at the appropriate cell density to reach confluence 72 h later. One well of untreated cells was seeded in low-serum (0.1% FBS) media as a positive control for senescence. The SA-β-gal assay was performed 96 h after transfection using the SA-β-gal assay kit (Cell Signalling Technology) according to the manufacturer’s instructions. Cells were imaged at 10x magnification using a Leica DMIL LED microscope. A total of 500 cells were counted from each well using the image analysis software Fiji [51 (link)].
4
Establishment of Induced Pluripotent Stem Cells
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Establishment of iPSCs was performed as reported before [10 (link)]. Cells were transferred to Matrigel® matrix (Corning) coated 6-well cell culture plates (Greiner Bio-One) and cultivated in mTeSR-1 (Stemcell Technologies). iPSC colonies were subcultured every seven days using 1U/ml Dispase (Stemcell Technologies).
5
Isolation and Expansion of PDLSCs
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PDLSCs were isolated from PDL, as previously described [29 (link)]. Briefly, the PDL strips were incubated for 70 min at 37 °C in a solution containing 2 mg/mL dispase (Gibco, Grand Island, NY, USA) and 2 mg/mL type I collagenase (Gibco, USA). The cell suspension was seeded in 6-well cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany). Cells were grown in DMEM-GlutaMAX growth medium (Gibco, USA) and supplemented with 15% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA), and 2 mM essential amino acids (Gibco, USA) at 37 °C and 5% CO2. Previously characterized PDLSCs at passages 3–5 were used in experiments [29 (link)].
6
hESC Maintenance and Passaging
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All hESC lines were maintained at 37°C and 5% CO2 in 6 well cell culture plates (Greiner) coated with 1% Matrigel hESC-Qualified Matrix (Corning) prepared in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Thermo Fisher Scientific). Cells were fed daily with Essential 8 medium (E8, Thermo Fisher Scientific) and passaged at 80% confluency using Versene solution (Thermo Fisher Scientific) for 1.5 minutes at 37°C followed by manual dissociation with a serological pipette. All cells were kept below passage 25 and confirmed as negative for mycoplasma infection.
7
Culturing A549 Cells for Experimentation
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The A549 cell line (DSMZ) was used in our experiments. Cells were cultured in DMEM High Glucose medium (PAA Laboratories GmbH) supplemented with 10 μg/ml gentamicin (Sigma-Aldrich) in 175-cm 2 flasks (Greiner Bio-One); 10% fetal calf serum (FCS; PAA) and HEPES buffer (PAA; S11-001) were added as well.
For the experiments, cells were harvested and counted, and 500,000 cells were seeded in 6-well cell culture plates (Greiner Bio-One). The cells were incubated overnight in 2 ml RPMI 1640 medium (PAA) supplemented with 5 ml of 200 m M L -glutamine (PAA), 5 ml of MEM NEAA (PAA) and 10% FCS. For the experiments, the medium was removed, and the experiments were carried out in RPMI 1640 medium with L -glutamine and NEAA. In all the experiments, incubation was always conducted at 37 ° C, 95% air and 5% CO 2 . As a negative control, in all the experiments, 1 well was pre-incubated with 900 μl of the medium only.
For the experiments, cells were harvested and counted, and 500,000 cells were seeded in 6-well cell culture plates (Greiner Bio-One). The cells were incubated overnight in 2 ml RPMI 1640 medium (PAA) supplemented with 5 ml of 200 m M L -glutamine (PAA), 5 ml of MEM NEAA (PAA) and 10% FCS. For the experiments, the medium was removed, and the experiments were carried out in RPMI 1640 medium with L -glutamine and NEAA. In all the experiments, incubation was always conducted at 37 ° C, 95% air and 5% CO 2 . As a negative control, in all the experiments, 1 well was pre-incubated with 900 μl of the medium only.
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