Chinese hamster lung (CHL) cells stably transfected with the human GLP1-receptor (CHL-hGLP1R)31 (link) are a donation from Martin Béhé (PSI, Switzerland). CHL-hGLP1R were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) GlutaMax (Gibco, Invitrogen, catalog 61965) supplemented with 10% heat-inactivated fetal calf serum (vol/vol), 100 units/ml penicillin and 100 μg/ml streptomycin, 50 mg/ml geneticin (G418) sulphate solution (PAA laboratories GmbH, GE Healthcare), 1 mM sodium pyruvate, and 0.1 mM Non-Essential Amino Acids (NEAA), in a humidified 5% CO2 atmosphere at 37 °C. hGLP1R negative CHL cells were grown in the same medium but without geneticin. The cells were harvested by trypsinization with trypsin/EDTA. CHL-hGLP1R and CHL negative cells were seeded on μ-Dish 35 mm (Ibidi) and cultured overnight. The cells were washed three times with Krebs buffer (NaCl 7.795 g/L, KCl 0.354 g/L, KH2PO4 0.162 g/L, MgS4H2O 0.293 g/L, CaCl2H2O 0.374 g/L, NaHCO3 0.424 g/L, Hepes 2.39 g/L) and incubated at 37 °C or 4 °C with Cy5.5-exendin-3 (0–100 nM) for 90 min in Krebs buffer with 3.9 mM glucose. Following incubation, cells were washed three times with Krebs buffer and put at 4 °C before imaging. Cells were imaged using the dfOCM/confocal fluorescence dual system (Fig. 2 ) to match the signals originating from cells and the Cy5.5-exendin-3 tracer.
μ dish 35 mm
Manufactured by Ibidi
Sourced in Germany
The μ-Dish 35 mm is a laboratory dish designed for cell culture applications. It provides a uniform and controlled environment for the growth and observation of cells. The dish has a standard 35 mm diameter and is made of high-quality materials suitable for cell culture purposes.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Culture insert 2 well in, by Ibidi (2 mentions)
Sulfo sanpah, by Thermo Fisher Scientific (2 mentions)
μ slide 8 well, by Ibidi (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Transwell, by Corning (1 mentions)
α mem, by Biowest (1 mentions)
Immunofluorescence application solution kit, by Cell Signaling Technology (1 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
El6000, by Leica Microsystems (1 mentions)
Orca flash4.0 v2, by Hamamatsu Photonics (1 mentions)
Las af software, by Leica Microsystems (1 mentions)
Dmi6000b, by Leica Microsystems (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Jc 1 probe, by Thermo Fisher Scientific (1 mentions)
Evos inverted fluorescence microscope, by Thermo Fisher Scientific (1 mentions)
Lsm 810, by Zeiss (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
35 protocols using μ dish 35 mm
1
Imaging of GLP1-receptor expressing cells
2
Migration Assays for IPFP-MSCs
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IPFP-MSCs were seeded into Culture-Insert 2 Well in μ-Dish 35mm (Ibidi) or onto cartilage surface, and cultured for 24 h. Culture-Insert was removed and scratches on cartilage were made in the plate using a P200 pipette tip. After washing with PBS three times, the culture medium was changed with DMEM/F12 supplemented with 0.9% NS or HA or PRP or TE (mixing ratio: 1:1). To analyze the migration of IPFP-MSCs, images were taken at 0, 24, and 48 h, and cells that crossed the baseline were counted for analysis.
Besides, 5 × 104 cells/well of IPFP-MSCs were seeded into the upper chamber of Transwell (Corning, 8 μm) filled with DMEM/F12. The lower chamber was filled with DMEM/F12 supplemented with 0.9% NS or HA or PRP or TE (mixing ratio: 1:1). After 24 h, cells in the upper surface of the membrane were removed with a cotton swab. Cells that migrated to the other side of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. These cells were imaged and counted in 6 random view fields.
Besides, 5 × 104 cells/well of IPFP-MSCs were seeded into the upper chamber of Transwell (Corning, 8 μm) filled with DMEM/F12. The lower chamber was filled with DMEM/F12 supplemented with 0.9% NS or HA or PRP or TE (mixing ratio: 1:1). After 24 h, cells in the upper surface of the membrane were removed with a cotton swab. Cells that migrated to the other side of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. These cells were imaged and counted in 6 random view fields.
3
Functionalization of PDMS Scaffolds for μTissue Anchoring
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To stabilize the anchorage of the growing μTissue on the edges of the PDMS scaffold, Fn was covalently bound to the surface of the PDMS scaffolds using a heterobifunctional cross-linker (Sulfo-SANPAH, 1 mg/ml; Thermo Fisher Scientific, USA), as described previously (43 (link)). Fn was isolated from human plasma, as previously described (83 (link)). The functionalized substrates were attached with optical glue NOA-61 (Norland Optical adhesive 61, Norland, USA) to an ibidi-treated polymer-bottom dish (μ-Dish 35 mm, no. 81158, ibidi) that had previously been passivated using poly(l -lysine)-graft-poly(ethylene glycol) (PLL-g-PEG, 0.1 mg/ml; SuSoS, Switzerland). After assembling the cell culture system, it was washed once with phosphate-buffered saline (PBS) and ultraviolet-treated for 15 min. Before cell seeding, the surface was equilibrated with α-minimum essential medium (αMEM, Biowest SAS) supplemented with 10% fetal bovine serum at 37°C for at least 1 hour.
4
Glucocorticoid Receptor Nuclear Translocation
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Fibroblasts were cultured on μ-dish 35 mm (ibidi GmbH, Planegg, Germany) for 24 h, then stimulated with DMSO (vehicle control), 5 μM G75, or 1 μM DEX for 24 h. Cells were fixed, permeabilized, and blocked using Immunofluorescence application solution kit (Cell Signaling Technology, Danvers, MA, USA) according to manufacturer’s instructions. Cells were treated with primary antibody GR (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and with secondary antibody (Alex Fluor 594, Cell Signaling Technology, Danvers, MA, USA) for 1 h at 22–25 °C. Cells were further stained with DAPI solution (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. Fluorescence microscopy was performed using a Nikon i2 U microscope (Tokyo, Japan). All images were processed in Nikon NIS-elements software (Ver. 4.0, Nikon, Tokyo, Japan). GR nuclear translocation was quantified as previously described by using Image J (NIH, Bethesda, ME, USA) [49 (link)]. Briefly, the percentage of the total corrected fluorescence of GR nuclear section per the total corrected fluorescence of total GR cellular section was calculated.
5
Live Imaging of DNA Damage Response
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Laser micro-irradiation experiments were essentially performed as described previously (45 (link)) with some modifications. Briefly, the cells were grown on plastic μ-Dish 35 mm (Ibidi) and transfected with the eGFP fusion protein expression plasmid 48 hours prior to imaging. The cells were presensitized by adding 10 μg/ml of Hoechst dye 33258 to the medium for 5 min at 37°C. The recruitment and the real-time follow-up of the protein of interest was carried out using a Confocal Leica SP5 system equipped with a 37°C heating chamber attached to a DMI6000 stand using 63×/1.4 objective of the PICT-IBiSA Orsay Imaging facility of Institut Curie. DNA photodamage were locally induced using a 2-photon laser set to minimal power at 810 nm and focused onto a single spot of constant size (176 nm) within the nucleus to generate a point of photodamage. When indicated, recruitment of the protein of interest was followed for 10 min using a 488 nm argon laser line. The fluorescent protein enrichment at the photodamage site was extracted with the ImageJ software using an in house developed macro (45 (link)). Experiments were performed at least three times for each protein of interest.
6
Imaging FRET Dynamics in Astrocytes
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Astrocytes plated on glass-bottom culture dishes (μ-Dish 35 mm, iBidi) and transfected with the FRET probes for RhoA (Raichu-EV-RhoA, ref pmid 12860967) or Src (KRas-Src-YPet, ref pmid 18799748) were imaged in an inverted epifluorescence microscope (DMI6000B, Leica Microsystems). The donor fluorescent protein was excited with a mercury lamp coupled to a light attenuator (EL6000, Leica Microsystems), and the emission of both donor and acceptor fluorescent proteins was acquired with a digital CMOS camera (4 × 4 binning, ORCA-Flash 4.0 V2, Hamamatsu Photonics). A 440–520 nm dichroic mirror (CG1, Leica Microsystems) was used together with appropriate emission and excitation filters mounted in external filter wheels (Fast Filter Wheels, Leica Microsystems). LAS AF software (Leica Microsystems) was used to control all modules. Raw images were background subtracted and time-lapse videos representing FRET ratio values (FRET/Donor or Donor/FRET) were generated. Regions of interest were drawn over cells and detailed analysis was performed to generate time-plots. Videos were converted to intensity modulated display mode using custom ImageJ macros and FRET channel as intensity modulator. Src kinase inhibitor (SKI-1) was added (200 nM) as a chemical inhibitor of SRC.
7
Mitochondrial Dysfunction Assessment in DGCR8-Depleted Cells
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Mitochondrial dysfunction in DGCR8-depleted cells was assessed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanide iodide (JC-1) probes (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells (3 × 104 or 1 × 104) were seeded in imaging dishes (μ-Dish 35 mm, high or μ-Slide 8 Well; Ibidi) in complete cell growth medium. siDGCR8 transfected cells with or without SOD2 overexpression were treated with 1 μM Hoechst (Thermo Fisher) and 10 μM JC-1 probes dissolved in dimethyl sulfoxide (DMSO) for 10 min at 37 °C. Fluorescence images were acquired using either an EVOS fluorescence inverted microscope (Thermo Fisher Scientific) or confocal microscopes (LSM-780 or LSM-810, Zeiss).
8
Quantifying Promoter-Driven Gene Expression
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To quantify promotor related reporter gene expression level and duration in proliferating cells, the transfected HEK 293, C2C12, Ab1167 and H9C2 cells were harvested on 4 consecutive days post transfection. Before harvesting, the nuclei were visualized by Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA; 1:1000) staining and the GFP expression among the respective promotors was documented by fluorescence microscopy (IX83 microscope, Olympus, Tokyo, Japan). To maintain cell viability, the cells were analysed using life cell imaging microscope incubator (OKOLAB USA Inc.; Ambridge, PA, USA) at 37°C and 5% CO2.
For cell harvesting the medium was removed, cells were washed with PBS, detached in PBS using a cell scraper and the cells were divided into three fractions. Cells were pelleted by centrifugation (300×G/3 min) and cell pellets were snap frozen in liquid nitrogen and stored at –20°C until further use. C2C12 and Ab1167 myotubes were either harvested as described above or directly processed on the 35 mm glass-bottom dishes (μ-Dish 35 mm; ibidi) for further immunocytochemical staining, see below.
For cell harvesting the medium was removed, cells were washed with PBS, detached in PBS using a cell scraper and the cells were divided into three fractions. Cells were pelleted by centrifugation (300×G/3 min) and cell pellets were snap frozen in liquid nitrogen and stored at –20°C until further use. C2C12 and Ab1167 myotubes were either harvested as described above or directly processed on the 35 mm glass-bottom dishes (μ-Dish 35 mm; ibidi) for further immunocytochemical staining, see below.
9
Cell Migration Assay by Radiation
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Cells were cultured in Culture-Insert 2 wells in μ-Dish 35 mm (Cat No: 81176, ibidi, Germany) with a defined 500 μm cell-free gap. The silicone inserts were carefully removed at 24 h after radiation exposure with doses ranging from 0.2 Gy to 5 Gy, and the dishes were observed under an inverted microscope (Nikon Instruments Inc., New York, USA). The images were taken with a DS-Fi3 Microscopy Camera with DS-L4 Tablet Interface (Nikon Instruments Inc., New York, USA). The gap distances were recorded at different time points. At 6 h after the removal of inserts, the results were used for comparison.
10
Germination Dynamics of Spores
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To test whether individual spores recovered from the glass vials were capable of germination and outgrowth, spores were resuspended in the glass vial with 40 μL sterile water. A drop (7 μL) of this suspension was applied to a cell culture dish with a thin plastic bottom (μ-dish 35 mm, ibidi GmbH, Germany) and dried for 20 minutes at ambient room temperature. Then, spores were covered with a thin (~ 1 mm thickness) layer of 1.5% LB-agar to initiate germination and imaged at 37°C in a temperature-controlled incubation system by an automated inverted light microscope (TE2000-E Eclipse, Nikon) using phase-contrast and a NA of 1.3. Images were recorded with a digital color CCD camera (DS-2Mv, Nikon) at a resolution of 1600x1200 Pixel (12 bit) and with 5 seconds interval.
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