To mimic native and stiffened mechanical environments of adult cardiac tissue29 (link),30 (link),75 (link), cell culture dishes were coated with soft or stiff polydimethylsiloxane (PDMS, Sylgard®527, Dow Corning) by using different mixing ratios (component A:B): 1:1 (E=12.7 ± 5.0 kPa) and 1:4 (E=139.7 ± 16.2 kPa). To enable live imaging at high magnification using a 100× objective, PDMS was deposited as thin layer (~100 µm) in gridded imaging dishes (µ-Dish 35 mm high Grid-500, ibidi). PDMS coated dishes were degassed under vacuum for 30 min, cured for 1h at 80°C, ozone-activated via corona arc-discharge (30s) and coated with reduced growth factor basement membrane matrix (Geltrex®, Gibco) for 1h at 37°C to provide attachment sites similar to the cardiac basement membrane. PDMS stiffness was determined via AFM using a spherical borosilicate glass tip (diameter=10 µm, stiffness=0.85 N/m), and Young’s modulus E was calculated using a Hertz contact model76 (link). Cells were also cultured on cell culture dishes coated with Geltrex basement membrane matrix but without PDMS coating to represent traditional experiment using tissue culture plastic (TCP).
Grid 500
Manufactured by Ibidi
Sourced in Germany
The Grid-500 is a laboratory equipment designed to provide a standardized grid pattern for various applications. It features a grid of 500 squares, each measuring 1 mm x 1 mm, for a total grid size of 50 mm x 50 mm. The Grid-500 is made of durable materials and can be used for a variety of purposes in scientific and research settings.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Sylgard 527, by Dow (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Cryostor cs10, by BioLife Solutions (1 mentions)
Cumate, by System Biosciences (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Fv1200, by Olympus (1 mentions)
μ dish, by Ibidi (1 mentions)
Eclipse ti, by Nikon (1 mentions)
7 protocols using grid 500
1
Engineered Cardiac Microenvironments
2
Cryopreservation of Human Mesenchymal Stem Cells
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For cryopreservation, hMSCs were seeded on culture-dishes (μ-Dish 35 mm, high Grid-500, ibidi, Matrinsried, Germany). Before freezing, culture medium was replaced by 500 μl Cryostor CS-10 (containing 10% DMSO, BioLife Solutions, Bothell, WA, USA). Cells were placed into a 4°C-precooled, programmable freezer (Ice-Cube 14S, SY-LAB, Neupurkersdorf, Austria) and frozen through temperature reduction at a cooling rate of 1°C/min or 10°C/min respectively. At -80°C, samples were exposed to liquid nitrogen (LN2) in order to mimic storage below the glass transition temperature of water for a few seconds. Upon thawing, 2ml of culture medium pre-warmed to 37°C was pipetted onto the cells. After washing with culture medium to remove DMSO, cells were incubated at 37°C, with 5% CO2 for recovery.
3
Inducible Cdc42-FLARE Cell Imaging
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Inducible Cdc42-FLARE cell line was seeded in six-well dishes 60 h before imaging at 50% confluency. At 48 h before imaging, the cells were induced with 300 µg/ml cumate (Systems Biosciences), and cumate concentration was maintained constant throughout the experiment. For protein depletion assays, 48 h before imaging, cells were transfected with RNAi using Oligofectamine. For protein expression, ∼18 h before imaging, cells were transiently transfected with ARHGAP10 constructs using Lipofectamine 3000 (Life Technologies) or the cerulean control using X-treme Gene 9. At 24 h before imaging, cells were transferred to a µDish35 mm, high with Grid-500 (81166; Ibidi), which was used for live imaging and locating the previously imaged cells after fixation.
4
Femtosecond Laser-Induced Mitochondrial Stimulation
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The Optical system was established on confocal microscopy (FV1200, Olympus) coupled with a femtosecond laser (BlueCut, Menlo, 1030 nm, 1 MHz, 220 fs). The laser was focused by an objective (water-immersed, ×60, 1.2 N.A.). Experiments were performed at 37 °C controlled by a mini-incubator on the microscope stage. Cells were localized by Petri dishes with a grid at the bottom (Grid-500, Ibidi).
The femtosecond-laser stimulation was delivered by the galvo-mirrors to the predefined region of the targeted mitochondrion, as demonstrated in Fig.1a . The laser exposure was controlled by a mechanical shutter that set for 0.1 s open, which was synchronized with the galvo-mirrors and only open when the femtosecond laser focus scanning in the region. The power of the femtosecond laser was about 8 mW measured at the specimen. The diameter of the laser focus point was approximately 0.7 μm. Images were recorded by confocal microscopy continuously before and after the stimulation. To take the real-time microscopy, the stimulation was set as a single frame of microscopy and inserted into the continuous confocal microscopy sequence at t = 5 s. In our experiments, each cell that was selected for UPLaS suffered only a single-time laser stimulation, during which only an individual mitochondrion was stimulated by the femtosecond laser.
The femtosecond-laser stimulation was delivered by the galvo-mirrors to the predefined region of the targeted mitochondrion, as demonstrated in Fig.
5
Imaging and Quantifying Tunneling Nanotubes
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We seeded PANC-1 cells at 1 × 105 cells/mL in a μ-Dish (35 mm, high Grid-500; ibidi GmbH, Gräfelfing, Germany) under normal cultivation conditions (5% CO2 at 37 °C) overnight. Then the cells were cultured in the macrophage-CM for another 48 h. The cells were washed with phosphate-buffered saline (PBS, pH = 7.4) three times and then fixed with 3.7% formalin for 15 min at room temperature. We removed the fixative and washed the cells with PBS three times, and then used 1 μg/mL DAPI (Thermo Fisher Scientific) to stain the nuclei. After 10 min of incubation, the fixed cells were thoroughly washed with PBS and then imaged with a 20× phase-contrast objective installed on an inverted microscope (ECLIPSE Ti, Nikon, Tokyo, Japan). We counted the TNTs following these two criteria: (1) the tube connected two cells, and (2) the tube length was longer than 50 μm.
6
Femtosecond laser cell viability
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To measure cell viability and proliferation rate after femtoSOC, dishes with a grid at the bottom (Grid-500, ibidi) were adopted to locate cells for femtoSOC. In brief, all cells within the grid of Grid-500 dishes were scanned by the femtosecond laser and then cultured at 37 °C in a humidified 5% CO2 atmosphere. After 6, 15, or 25 h, cells were stained with PI and observed by the confocal microscope to record the number of viable and dead cells.
7
Anti-estrogen and Hormone Treatments on Breast Cancer Cells
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MMTV-PyMT WT or BK KO, MDA-MB-157, MDA-MB-453, or MCF-7 cells were maintained for 24 h in duplicate in Grid-500 μ-dishes for anti-oestrogen/hormone treatments or in μ-slides 8 Well Grid-500 for siRNA experiments (ibidi, Planegg, Germany). Following serum starvation for 72 h, re-stimulation with serumcontaining media for 72 h was performed in the absence or presence of anti-oestrogens or hormones at various concentrations. siRNAmediated knockdown (KD) of BKα and BKγ1 was performed at the first day of serum starvation and repeated at the first day of re-stimulation. Cell counts were determined in defined areas of interest using digital images taken every 24 h for siRNA and growth factors experiments (time points: 0, 24, 48, and 72 h) and at the beginning and the end during the 72-h period of anti-oestrogen/hormone treatments. were estimated according to Barry and Lynch (1991) (link), and data were corrected off-line for the estimated liquid junction potentials.
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