Staphylococcus aureus 04-02981 was grown in TSB overnight at 37°C, 150 rpm, then it was diluted to an OD600 of 0.05 and further incubated at 37°C for 4 h (mid-exponential phase, OD600 ∼1.5). Then, the culture was transferred to a μ-Dish (μ-Dish 35 mm, low, from ibidi GmbH, Martinsried, Germany) containing Ag, or AGXX® (sheet surface: medium volume ratio = 0.8) and incubated at 37°C for 24 h. The culture was removed from the μ-Dish, and the biofilm on the μ-Dish was washed three times with phosphate buffered saline (PBS). The biofilm was stained for 10 min in the dark with Hoechst 33342 (5 μg/mL) and propidium iodide (1 μg/mL) (Thermo Fisher, Eugene, OR, United States). The staining solution was then replaced with 50% glycerol to prevent movement of bacteria during imaging. Imaging was performed with a Nikon TiE-based Visitron spinning disk confocal microscope using a 100× NA1.45 objective. Fluorescent dyes were excited using 405 nm (Hoechst 33342) and 561 nm (propidium iodide) laser lines and fluorescent emission captured through appropriate filters onto an iXon888 EMCCD detector (Andor, Belfast, United Kingdom). Images were subsequently analyzed using Fiji (ImageJ) version 3.2.0.2.
μ dish
Manufactured by Ibidi
Sourced in Germany
The μ-Dish is a cell culture dish designed for high-resolution microscopy. It features a thin glass bottom that allows for optimal imaging conditions.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Cell 40 μm pore size cell strainer, by Corning (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lsm 800 microscope, by Zeiss (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Hepes, by Harvard Bioscience (1 mentions)
Dulbecco modified eagle medium (dmem), by Harvard Bioscience (1 mentions)
75 protocols using μ dish
1
Quantifying Staphylococcus aureus Biofilm Formation
2
Migration Assay for A549 Cells
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This method for analysis of migration was conducted as described previously31 (link). Briefly, 5 × 103 of each of serum-starved A549luc stable cell lines were seeded into the μ-Dish (35 mm high, purchased from IBIDI) and cultured overnight for adhering before a clear area was created by removing the Culture-Insert from the μ-Dish. Photos were taken at the indicated time.
3
Quantifying Cell Migration and Invasion
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Scratch wound migration assays were performed on H1299 V and KD cells as previously described.10 (link) For A549 cells, a 35 mm μ-Dish (Ibidi) was used with complete media containing EGF (50 ng/ml) for 9 hours. For live cell migration assays, H1299 V and KD1 cells were grown to confluence in either side of a 35 mm μ-Dish (Ibidi), serum starved overnight, and culture insert removed prior to imaging on a Quorum WaveFX-X1 spinning disc confocal microscope (Quorum Technologies Inc.). Images were captured every 15 minutes for 18 hours. Videos were created using MetaMorph Microscopy Automation & Image Analysis Software (Molecular Devices). The migration distance of individual cells was measured using Image J software with Manual Tracker and Click Forward add-ons. Transwell invasion assays were performed as previously described.10 (link) A549 cells were treated with TGF-β1 (2 ng/ml) prior to these assays to enhance their invasiveness. After 48 hrs, DAPI-stained cells were imaged by epifluorescence microscopy (4 fields/insert) and scored using Image Pro Plus.
4
Cell Seeding on Micropatterned Surfaces
5
Cell Seeding on Micropatterned Surfaces
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Non-patterned grids were plasma cleaned. Cells were detached from cell culture flasks using 0.05 % trypsin-EDTA and seeded on pre-treated (either patterned or non-patterned) grids in glass bottom ibidi μ-Dish 35 mm high (ibidi, Martinsried, Germany).
Cells were seeded on fibronectin micropatterned surfaces right after being passed
through a cell 40 μm pore-size cell strainer (Corning, Amsterdam,
Netherlands) at a density of 2 × 104 cells/cm2 for
HeLa and 8 × 103 cells/cm2 for RPE1 cell lines.
After seeding, grids were incubated for 1.5-2 h for HeLa cells or 20-35 min for
RPE1 cells. Next, grids were transferred to a new cell-free dish and incubated
at 37°C with 5 % CO2 to allow cell adhesion to the grids.
Transfer to a new dish was beneficial to remove cells that were non-specifically
attached to areas outside the patterns. Cells were vitrified 4-6 h post-transfer
for RPE1 cells (to attain a higher number of grid squares with single cells) or
after overnight incubation for HeLa cells.
At least 60 grids have been seeded with either HeLa or RPE1 cells obtaining reproducible results with cells settling and adhering to the micropatterned areas.
Cells were seeded on fibronectin micropatterned surfaces right after being passed
through a cell 40 μm pore-size cell strainer (Corning, Amsterdam,
Netherlands) at a density of 2 × 104 cells/cm2 for
HeLa and 8 × 103 cells/cm2 for RPE1 cell lines.
After seeding, grids were incubated for 1.5-2 h for HeLa cells or 20-35 min for
RPE1 cells. Next, grids were transferred to a new cell-free dish and incubated
at 37°C with 5 % CO2 to allow cell adhesion to the grids.
Transfer to a new dish was beneficial to remove cells that were non-specifically
attached to areas outside the patterns. Cells were vitrified 4-6 h post-transfer
for RPE1 cells (to attain a higher number of grid squares with single cells) or
after overnight incubation for HeLa cells.
At least 60 grids have been seeded with either HeLa or RPE1 cells obtaining reproducible results with cells settling and adhering to the micropatterned areas.
6
Digital Holographic Video Microscopy for Analyzing ST-T1b Cell Migration
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ST-T1b cell migration and motility were analysed via digitalholographic video microscopy (DHVM) (Greve et al., 2012b; Kemper and von Bally, 2008) . 50,000 ST-T1b cells per well were cultured in six-well-plates for 24 h prior to miRNA transfection. After further 24 h, transfected ST-T1b cells were transferred to Petri dishes (ibidi μ-Dish, ibidi GmbH, Munich, Germany) and the medium was exchanged to DMEM + 10% FCS + 20 mM HEPES (Biochrom). Cells then were observed for 48 h by quantitative phase contrast imaging at 37°C utilizing an inverted microscope (iMIC, Till Photonics, Gräfelfing, Germany) with an attached DHVM module (Kemper et al., 2006) and were subsequently analysed by custom-built software for automated cell tracking as reported previously (Kemper et al., 2010) . In order to quantify cell motility from the resulting migration trajectories the mean squared displacement was calculated as described in Sridharan et al. (2011) .
7
Imaging Transcription Bodies in Live Zebrafish Embryos
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For imaging on a spinning disk microscope, dechorionated embryos were mounted at 32-cell stage in small droplets of 1% low-melting agarose (Invitrogen, catalogue number 16520-050) in 75% v/v 0.3× Danieau’s solution and 25% v/v Optiprep Density Gradient Medium (Sigma-Aldrich, catalogue number D1556)68 (link) on a Ibidi μ-Dish 35 mm high (catalogue number 81158). Embryos were brought closer to the coverslip surface by keeping the dish upside down until the agarose solidified (~20 min). More agarose was then added to avoid drying out. Transcription bodies in live embryos were imaged on a Nikon eclipse Ti2 with a Yokogava CSU-W1 Spinning Disk unit at 28 °C (Okolab temperature control and microscope enclosure). Multiposition, z-stack (20 μm in 41 steps, NIDAQ Piezo Z) timelapse (2 min time interval) imaging was performed with a Nikon ×60/1.2 Plan Apochromat VC water objective. Images were captured in parallel on two Photometrics Prime 95B cameras with 100 ms exposure time at 10% laser intensity.
8
Characterization of gold nanoparticle uptake
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Absorption spectra were recorded using a Jasco V-670 UV-Vis-NIR spectrometer with a slit width of 2 and 1 nm spectral resolution. Particle size distribution and zeta potential were measured by a Zetasizer NanoZS90 instrument (Malvern Instruments). Analysis was performed at a scattering angle of 90° and temperature of 25 °C. Dark field images of OCI-AML3 and THP1 cells incubated with GNP-MDS-Pl were acquired using an inverted Zeiss Axio Observer Z1 microscope. To capture the cells, a few microliters of cell suspension were dropped onto an Ibidi μ-Dish and enclosed with a cover slip. A 100-W halogen lamp was used for illumination which was focused on the sample using a high numerical immersion condenser (NA = 1.4), and the scattered light was collected by an LD Plan-Neofluar ×20 objective (NA = 0.4, Zeiss).
9
Cellular Uptake of Microcapsules
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Cells were seeded in a 35 mm μ-Dish (Ibidi, Germany) and cultured in an atmosphere containing 5% CO2, at 37 °C. Six hours after cell seeding, the medium was replaced with the medium containing the microcapsules in the amount of 20 capsules per cell. After 24 h, cells were washed three times with PBS, and fluorescent microphotographs were made using Axiovert 200 (Zeiss, Germany). Cell nuclei were stained with Hoechst 33342, and mitochondria were stained with MitoTracker® Green FM (Thermo Fisher Scientific).
10
Primary Rat Hippocampal Neuron Culture
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Dissociated hippocampal neuronal cultures were prepared from E18 rat embryos and cultured according to the Banker protocol. All experiments involving neurons were carried out at 16–21 days in vitro. For confocal imaging, cells were plated onto 13 mm round glass coverslips (thickness 1.0) placed in 35 mm Petri dishes (4/dish). For 3B imaging, cell were plated onto either 22 mm square glass coverslips (Carl Zeiss, Germany) or 35 mm μ-Dish (Ibidi, Germany), both thickness 1.5. To reduce variability, each experiment was carried out using sister cultures, i.e. cultures originating from the same preparation and cultured identically; to further enhance reproducibility, each confocal imaging experiment was carried out using the coverslips cultured within the same Petri dish.
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