A Nikon A1-MP confocal microscope outfitted with a 2-photon Ti:Sapphire laser (Mai Tai, Spectra Physics, Newport Beach, CA, USA), emitting 80-fs pulses at a repetition rate of 80 MHz, was used to characterize the functional properties of ARPE-19 cells. An on-stage incubator (OKOLAB) kept a constant temperature of 37 °C and a 5% level of CO2. For the quantification of intracellular non-polar aggregates, the lipophilic probe Laurdan was used. Cells were treated with 1 µM of Laurdan. Laurdan intensity images (excitation: 740 nm) were recorded in the two emission ranges 450/50 nm and 525/50 nm with a 1024 × 1024-pixel resolution, and a 60× oil-immersion objective was used to visualize lipid aggregates.
On stage incubator
Manufactured by Okolab
Sourced in Italy
The On-stage incubator is a laboratory equipment designed to maintain a controlled environment for samples during microscopy observation. It provides temperature regulation to ensure sample viability.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
A1 mp confocal microscope, by Spectra-Physics (1 mentions)
Perfect focus system, by Nikon (1 mentions)
Nis software, by Nikon (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Imagej, by Leica (1 mentions)
Imaris, by Leica (1 mentions)
Chameleon vision 2, by Coherent Inc (1 mentions)
Csu x1, by Yokogawa (1 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (1 mentions)
6 protocols using on stage incubator
1
Quantifying Intracellular Lipid Aggregates
2
TIRF Microscopy for Single Particle Tracking
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SPT measurements were performed with an inverted microscope (Ti-Eclipse, Nikon Instrument, Japan) adapted with an × 100 NA = 1, 49 TIRF oil immersion objective and an appropriate dichroic 405/488/561/635 nm (ref Di03-R405/488/561/635 SEMROCK, USA). The excitation laser (488 nm Argon LASER, Melles-Griot, USA and 561 nm, OXXIUS, France) was injected into the TIRF Nikon module into a mono-mode fiber and focused on the backplane of the objective. Illumination was performed in HILO mode29 (link) to improve contrast in images. HILO sheet thickness was estimated to 4–6 µm29 (link). Perfect Focus System (PFS, Nikon) was set to avoid drift on Z-axis (defocusing) on the objective, relative to the coverslip. Experiments were acquired under continuous illumination (488 nm 20 kW/cm2 on the sample, or 561 nm 20 kW/cm2 on the sample). Values of illumination are derived for a maximal power from the laser of about 100mW for a field of view with a diameter of around 25 µm. Emitted fluorescence was harvested on an sCMOS camera (PCO EDGE 4.2, PCO, Germany) with a pixel size of 6,5 µm. The acquisition format was 100 × 100 px, and the acquisition rate was set at 100 Hz (10 ms per frame). All devices were driven by NIS software (Nikon Instruments, Japan). A thermostatic chamber (‘on-stage-incubator’, OKOLAB, Italy) was used to keep samples at 37 Celsius degrees.
3
Visualizing 3D Tumor Cell Invasion
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For time-lapse experiments, CAFs were stained with a lyophilic carbocyanine dye (Vybrant DiI-Cell Labeling Solutions; ThermoFisher) according to the manufacturer’s recommendation. Cells were embedded in collagen as described in the Invasion assay section. The dish was incubated at 5% CO2 and 37°C in the on-stage incubator (Okolab). For fixed and live 3D samples, images were acquired with an inverted AOBS two-photon laser-scanning confocal microscope (SP8; Leica) coupled with a femtosecond laser (Chameleon Vision II; Coherent Inc.) using a 25×/1.0 NA water immersion objective. The microscope was equipped with three nondescanned HyD detectors: NDD1 (500–550 nm), NDD2 (≥590 nm), and NDD3 (405 nm). Fluorescence channels were recorded simultaneously using the excitation wavelength 980 nm. Collagen was visualized by either second harmonic generation using the excitation wavelength 910 nm or by confocal reflectance microscopy that does not interfere with DAPI staining, using light at a wavelength of 488 nm and a standard photomultiplier tube detector at a low gain (500 V). Images were recorded every 10 min up to 72 h. 3D stacks were obtained at a step size of 2-µm intervals. The images were processed with the Leica Application Suite, ImageJ, and Imaris (Bitplane).
4
Live Cell Imaging of Genome-Edited SUM159 Cells
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Live cell imaging of genome edited SUM159 AP-2-σ2-EGFP cells seeded on arrays of 1D nanopillars with D ~ 600 nm was performed using a spinning disk confocal microscopy (Nikon TI Andor CSU-X1 equipped with a Yokogawa spinning disk unit (Andor)) (MRI facility, Montpellier) equipped with a 488 laser beam (60 mW). Acquisitions were performed with a 60x/1.4 objective. During imaging, cells were maintained at 37°C, 5%CO2 in an onstage incubator (Okolab). Movies were recorded with a mono dichroic mirror (488nm) and a GFP emission filter (525-30nm). Samples were exposed to laser light for 100ms every 2s for 5 min and images were acquired using an EMCCD iXon897 (Andor) camera. A 0.15μm z-step was used to cover all the nanopillars length in every z-stack acquisition.
5
Fluorescent Dextran Perfusion Assay
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The 10 kDa Dextran labeled with AlexaFluor568
(Thermo Fisher Scientific) was diluted in HUVEC (EndoGRO) medium at
a final concentration of 1 μg/mL. The solution was perfused
into the blood vessel model channel at a flow rate of 5 μL/min
using the syringe pump. The perfusion of dextran was monitored using
Nikon Eclipse Ti2 epifluorescent microscope. The chip was placed in
the on-stage incubator (OKOlab) at a temperature of 37 °C and
5% CO2, the perfused fluorophore was excited at 525 nm,
and the emission was collected at 650 nm.
(Thermo Fisher Scientific) was diluted in HUVEC (EndoGRO) medium at
a final concentration of 1 μg/mL. The solution was perfused
into the blood vessel model channel at a flow rate of 5 μL/min
using the syringe pump. The perfusion of dextran was monitored using
Nikon Eclipse Ti2 epifluorescent microscope. The chip was placed in
the on-stage incubator (OKOlab) at a temperature of 37 °C and
5% CO2, the perfused fluorophore was excited at 525 nm,
and the emission was collected at 650 nm.
6
Kidney Organoid Formation and Imaging
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Embryonic kidneys were dissected from embryonic day 11.5 mouse embryos from crossing of Wnt4Cre (Shan et al., 2010) (link) and tomato floxed Rosa26 Green fluorescent protein (GFP; mT/mG) reporter mice (Muzumdar et al., 2007) (link) as described in (Junttila et al., 2015) (link). Intact UBs were treated with GDNF and dissociated MM with BMP7 and FGF2 as in (Junttila et al., 2015) (link). The intact UB was reaggregated with MM cells and incubated overnight to form a kidney organoid. The organoids were set to grow in a FiZD culture (Saarela et al., 2017) (link) for time-lapse imaging into a temperature and gas controlled on-stage incubator (OkoLab, Italy) on a Zeiss LSM780 confocal microscope.
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