Cy3-(GT)6-SWCNTs and Cy3-(GT)30-SWCNTs were first filtered three times using 100 kDa Amicon centrifuge filters (Millipore) to remove free Cy3-DNA from solution. The cells were seeded onto 35 mm glass-bottom Petri dishes (MatTek) to a final concentration of 500 000 cells/dish and allowed to culture overnight in an incubator. The media was removed from each well, replaced with 1 mg/L of filtered Cy3-(GT)6-SWCNTs or Cy3-(GT)30-SWCNTs diluted in media and incubated for 30 min to allow internalization into the cells. The SWCNT-containing media was removed, the cells were rinsed three times with sterile PBS (Gibco), and fresh media was replenished for each sample. The Petri dishes were mounted in a stage incubator (Okolab) on an Olympus IX-73 inverted microscope with a UApo N 100×/1.49 oil immersion objective for epifluorescence imaging with a U-HGLGPS excitation source (Olympus) filtered through a Cy3 filter cube. The fluorescence images were analyzed by extracting average fluorescence intensity values of individual cell ROIs using ImageJ.
Stage incubator
Manufactured by Okolab
Sourced in Italy
The Stage Incubator is a piece of lab equipment designed to provide a controlled environment for live cell imaging and microscopy applications. It maintains a stable temperature, humidity, and gas composition within a localized area surrounding a microscope stage.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements software, by Nikon (2 mentions)
Digital camera, by Nikon (2 mentions)
Tcs sp8 x confocal microscope, by Leica (2 mentions)
Amicon centrifuge filter, by Merck Group (1 mentions)
Uapo n 100 1, by Olympus (1 mentions)
Sterile pbs, by Thermo Fisher Scientific (1 mentions)
35 mm glass bottom petri dishes, by MatTek (1 mentions)
Ix73 inverted microscope, by Olympus (1 mentions)
Tcs sp8x laser, by Leica (1 mentions)
Hc plapo 63x oil immersion objective, by Leica (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Phase contrast microscope, by Nikon (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
Eclipse ti e ds qi2 microscope, by Nikon (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
12 protocols using stage incubator
1
Quantifying Cellular Uptake of Cy3-SWCNTs
2
Time-lapse 3D Confocal Imaging of Cytoskeleton
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Confocal optical sectioning is performed with on a Leica TCS-SP8X laser-scanning confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with a white light laser (WLL) source and a 405nm diode laser. Sequential confocal images are acquired using a HC PLAPO 63x oil-immersion objective (1.40 numerical aperture, Leica Microsystems). Z-reconstructions of serial single optical sections are acquired every 3 min (for SiR-Actin probe) or 5 min (for SiR-Tubulin probe), and carried out with a 512x512 format, scan speed of 400Hz, a pixel size of 0,3 μm, and z-step size of 0,5 μm. Lasers’ power, beam splitters, filter settings, pinhole diameters and scan mode are the same for all examined samples of each staining. Time-lapse microscopy was performed with a stage incubator (OkoLab, Naples, Italy) allowing to maintain stable conditions of temperature, CO2 and humidity during live-cell imaging.
Z-reconstructions are imported into Imaris (Bitplane, Zurich, Switzerland) software to obtain their three-dimensional (3D) surface rendering. To improve contrast and resolution of confocal raw images, deconvolution analysis (3D Deconvolution software, Leica Microsystems) is applied to Z stacks before 3D reconstruction. The reconstructed images were assembled in Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA).
Z-reconstructions are imported into Imaris (Bitplane, Zurich, Switzerland) software to obtain their three-dimensional (3D) surface rendering. To improve contrast and resolution of confocal raw images, deconvolution analysis (3D Deconvolution software, Leica Microsystems) is applied to Z stacks before 3D reconstruction. The reconstructed images were assembled in Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA).
3
CTL-target cytotoxicity imaging protocol
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24 h before imaging and 5–8 d after stimulation, 5 × 106 CTLs were transfected with 5–16 µg DNA using the Mouse CD8 T cell nucleofection kit (Amaxa; Cat# VPA-1006). EL4s expressing either Farnesyl-5-TagBFP2 or Mem-RFP670 were used as targets and pulsed for 30 min with 1 µM peptide at 37°C and 8% CO2 before washing into serum-free DMEM and applying to 1 µg/ml murine ICAM-1–coated 35-mm glass-bottomed culture dishes at 6.5 × 105/ml. After 5 min to adhere, unbound targets were washed clear with phenol-red free T cell medium plus 25 mM Hepes, and the dish was loaded onto the microscope. Approximately 2 × 106 nucleofected CTLs were dropped onto the dish, and imaging began within 5 min.
Imaging of CTL:Target interactions used the system described above with an Olympus Universal Plan Super Apochromat 60× 1.3 NA silicone oil objective and an OKOLAB stage incubator to maintain a 37°C temperature and ∼5% CO2 atmosphere. Each z-plane was separated by 0.8 µm with the z-dimension ranging from 16–20 µm and image stacks taken every 5–20 s for 20–40 min. Fluorophores were excited with 405-, 488-, 561-, and 640-nm lasers in each plane. Data were captured with the iQ3 software (Andor) before visualizing and analyzing with Imaris (Bitplane).
Imaging of CTL:Target interactions used the system described above with an Olympus Universal Plan Super Apochromat 60× 1.3 NA silicone oil objective and an OKOLAB stage incubator to maintain a 37°C temperature and ∼5% CO2 atmosphere. Each z-plane was separated by 0.8 µm with the z-dimension ranging from 16–20 µm and image stacks taken every 5–20 s for 20–40 min. Fluorophores were excited with 405-, 488-, 561-, and 640-nm lasers in each plane. Data were captured with the iQ3 software (Andor) before visualizing and analyzing with Imaris (Bitplane).
4
Calcium Signaling in Differentiated Cells
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In this work we followed the methodology proposed by Glaser et al. (2016) [72 (link)]. The differentiated cells were grown on 35 mm optical plates (Cod. 81156, Ibidi, Gräfelfing, Germany), coated with Matrigel, and washed with HBSS (Cod. 14025-050, Gibco, Carlsbad, CA, USA). The solution containing the Fluo-4 probe (Cod. F10489, ThermoFisher Scientific) was added and, after 15 min incubation, recording begun. After 3 min, 5 μM of ionomycin (Cod. I24222, Invitrogen, Waltham, MA, USA) was added to the cells to record the maximum fluorescence peak. The addition of 30 mM EGTA followed (Cod. SLBR7504V, Sigma Aldrich). Recording ended after 10 min. Live recording was conducted with a frame rate of 2 fs/sec, magnification 20× with a 1024 × 800 format and an electronic zoom at 2.0 using the SP8X Leica confocal microscope equipped with a resonant scanner for fast imaging (8.0 MHz), and a stage incubator (OkoLab, Italy) allowing maintenance at 37 °C and a humidified atmosphere with 5% CO2. For each biological replicate, 10 to 15 cells were measured. The changes in fluorescence intensity over time were represented in the curve graph reported.
5
Fibroblast Migration Assay Protocol
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1.6 × 105 cells/35 mm dish were plated to create a confluent monolayer. Dishes were cultured for approximately 48 hours at 37°C to allow cells to adhere and spread. Cell monolayer was scratched in a straight line with a p10 sterile pipette tip. Fresh medium was pipetted in the dish, after one wash with medium to remove debris. Culture plates were then placed under a phase‐contrast microscope (Nikon, Tokyo, Japan) equipped with stage incubator (Okolab, Pozzuoli, Italy), and the migration of fibroblasts at both edges of the wound was documented acquiring one picture every 10 minutes for 12 hours by digital camera (Nikon). Data were analysed by NIS Elements software (Nikon) and expressed as mean speed of migration ± SE.
6
Wound Healing Assay with HaCaT Cells
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HaCaT cells were seeded at a density of 2 × 105 cells/well in a 12-well plate coated with collagen using DMEM containing 10% w/v FBS to obtain a monolayer covering 90–100% of the well area in 24 h [10 (link)]. Then, the medium was removed and the monolayers were scratched using a sterile tip as previously described [10 (link)]. Then, after rinsing with sterile PBS 3 times, media depleted of FBS was added, to resemble the starvation conditions used in flask cultures for proteomic analyses. Three wells were added with TVE 5% v/v, while no additional ingredient was present in control wells. The multiwell was placed on a stage incubator (OKOlab S.r.L., Naples, Italy), and the experiment was followed by the time-lapse video microscopy station for 24 h.
7
Live FRET Imaging of EGF Signaling
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Live FRET changes induced by bath stimulation with recombinant EGF (1 mg/ml) were assessed in embryos mounted in 1.5% low-melting agarose dissolved in E3 medium. Embryos are imaged using TCS-SP8X confocal microscope (Leica Microsystems) equipped of a stage incubator (OkoLab, Italy) under controlled conditions (temperature of 28 °C and a humidified atmosphere), using a simple time lapse mode (x,y,z,t) scanning mode in the “Calcium calculator” wizard with 1024 × 1024 image format at 400 Hz, a 5 μm z-step size and with a time interval of 1 min. The 458 nm laser line (50% of the Argon laser power) was used to excite the CFP donor, and the detection of the two wavelength ranges, one for CFP (465–500 nm) and one for YPet (525–570 nm) emission spectra, respectively.
8
Live Imaging of ARF3 Dynamics
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For live imaging, COS-1 cells (10 × 104) were seeded into μ-dishes 35 mm (Ibidi) 24 h before transfection. The day after, cells were co-transfected with WT or mutant mCherry-tagged ARF3 and EGFP-GalT constructs. Four hours post-transfection, time-lapse acquisitions were performed with a Leica TCS-SP8X confocal microscope (Leica Microsystems) with a PlApo CS2 ×20/0.75 objective, using excitation lines at 488 nm (for EGFP, emission range 500–550 nm) and 594 nm (for mCherry, emission range 600–650 nm). Parallel live imaging of control and mutant samples was performed simultaneously using the Mark & Find mode of the LAS X software v.3.5. Cells were monitored every 15 min and imaging was carried out with a 1024 × 1024 format, 0.38 μm pixel size, scan speed of 600 Hz, a zoom magnification up to 1.5 and z-step size of 0.7 μm, time-lapse microscopy was performed with a stage incubator (OkoLab) allowing to maintain stable conditions of temperature at 37 °C, with 5% of CO2 and humidity during live cell imaging.
9
Live Imaging of BDNF-Stimulated Neuron Differentiation
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Before imaging, cells were labelled with Janelia Fluor JF646 Halo-Tag (kindly provided by Dr Luke Laevis, Janelia Laboratories, Ashburn, USA). For the BDNF stimulation experiments 1 pM and for the differentiation experiment 10 pM (increased concentration to account for the lower transfection efficiency) of the JF646 Halo-Tag ligand (HTL) were used, respectively. After labelling for 15 min at 37°C and 5% CO2, we washed the cells 3 times for 10 min with NMEM/B27 medium, supplied with 5 µg ml−1 gentamicin. We used 5 div (days in vitro) cultured hippocampal neurons for cell stimulation experiments (figure 1 ) and added 10 ng ml−1 BDNF (Preprotech, 10 µg) to the medium before imaging. The cells were measured for a maximum of 80 min and a stage incubator (Okolab, Ottaviano, Italy) controlled the temperature to 37°C and the CO2 content to 5%. For differentiation analysis, we used neurons cultivated in vitro for 5 h (stage 1), 1 day (stage 2), 3 days (stage 3), 7 days (stage 4), respectively.
10
Cell Migration Assay for CPC-P
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To evaluate the speed of migration of CPC-P cultured with EV-CM and of CPC-P of control group, scratch wound assay was performed as previously described (Castaldo et al., 2013 (link)). Briefly, cells were grown to confluence and a thin scratch was produced in straight line on culture plates with a 10 µl sterile pipette tip, leaving a cell-free zone. Cell culture dishes were first washed with medium to remove debris and then fresh medium was pipetted in the dishes. Plates were placed under Nikon Eclipse Ti-E DS-Qi2 Microscope (Nikon) equipped with stage incubator (Okolab, Pozzuoli, Italy) and the migration was documented acquiring pictures every 10 min for 8 h by digital camera (Nikon). Data were analyzed by NIS Elements software (Nikon) and expressed as mean speed of migration ± SEM.
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