The primary neuroglial culture was obtained as described above and divided into two groups 4 h after the cells were attached. First group was treated by MSC-EV at a concentration of 2.4 × 108 particles/mL, while the control group was treated with vehicle and the culture medium was not changed during the entire experiment. The analysis of cell cultures was carried out daily for 2 plates from each group during 7 days, after which they were not used. Hippocampal cell cultures were loaded with a Calcein-AM probe at a final concentration of 5 µM for 40 min at 37 °C in HBSS with 10 mM HEPES followed by a triple wash with HBSS. Cell slips were mounted in an experimental chamber and observed using a Leica DMI6000B fluorescent inverted motorized microscope with a HAMAMATSU C9100 high-speed monochrome CCD camera. For excitation and recording of Calcein fluorescence, a L5 filter set (Leica, Germany) was used, containing excitation filter BP480/40, beam splitter FT-505 and emission filter BP527/30, with a Leica EL6000 excitation light source containing a HBO 103 W/2 high-pressure mercury lamp. Neurite length analysis was performed using the Image J software.
C9100
Manufactured by Hamamatsu Photonics
Sourced in Japan
The C9100 is a high-speed, scientific-grade CCD camera developed by Hamamatsu Photonics. It features a large sensor size and high quantum efficiency for low-light imaging applications. The core function of the C9100 is to capture and record high-quality images and data with excellent sensitivity and speed.
Automatically generated - may contain errors
Lab products found in correlation
Leica el6000, by Leica camera (5 mentions)
Leica dmi6000b, by Leica camera (5 mentions)
Fu 2 filter set, by Leica camera (4 mentions)
Csu22, by Yokogawa (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Dmi6000b, by Leica camera (2 mentions)
Eclipse ti, by Nikon (2 mentions)
Volocity software, by PerkinElmer (2 mentions)
Ultraview vox, by PerkinElmer (2 mentions)
Fluoview software, by Olympus (2 mentions)
Eclipse ti e, by Nikon (2 mentions)
Em ccd, by Hamamatsu Photonics (2 mentions)
Scanr high content microscope, by Olympus (2 mentions)
Mt20 illumination system, by Hamamatsu Photonics (2 mentions)
Axiovert 200, by Yokogawa (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
El6000, by Leica camera (1 mentions)
L5 filter set, by Leica camera (1 mentions)
Wasabi software, by Hamamatsu Photonics (1 mentions)
36 protocols using c9100
1
Neuroglial Culture Evaluation with MSC-EVs
2
Fluorescent Bilayer Imaging and FRAP
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Imaging of fluorescent spherical supported bilayers containing 0.1% LRPE – 2% PtdIns(4,5)P2 – 97.9% DOPC was performed at room temperature using a 561-nm laser on a spinning-disk confocal (UltraVIEW VoX; PerkinElmer) with a microscope (Eclipse Ti; Nikon) with the Perfect Focus System using an Apochromat 100×, 1.49 NA oil immersion objective (Nikon). Digital images were acquired with an EM charge-coupled device camera (C9100; Hamamatsu Photonics) using Volocity software (PerkinElmer). Z-stacks were collected in steps of 0.1 μm and exposure time of 100 ms. Photobleaching and fluorescence recovery experiments were done as follows: (1) Recorded for 5s at 18.5s fr/s before photobleaching, (2) photobleached for ~4s and (3) recorded recovery of fluorescence for 60 s at a speed of 18.5 fr/s.
3
Fluorescence Imaging of LipoParticles
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Observation of the LipoParticles was performed on a widefield fluorescence microscope Leica DMI6000B using an excitation source Leica Halogen Bulb and an EMCCD camera (Hamamatsu C9100, 512 × 512 pixels, pixel size: 16 µm). A 100x oil immersion objective lens (HCX PL APO Leica, NA = 1.46) was used for both brightfield and fluorescence. A BGR filter (450/90, 502/15, 590/20) was used for fluorescence. On the glass coverslip (thickness 170 ± 5 µm), 10 µL of sample and 2 µL of a 5 M NaCl solution were mixed, to decrease the Debye length and favour adhesion of the charged LipoParticles onto the coverslip. The images were acquired with the LASAF software (Leica) to manage the microscope and the Wasabi software (Hamamatsu) to pilot the EMCCD camera. Images were processed using the ImageJ freeware.
4
Measuring Intracellular Calcium Dynamics
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To measure the changes in [Ca2+]i, cell cultures were stained with Fura-2 (4 µM; 40 min incubation; 37 °C) in Hank’s balanced salt solution (HBSS) composed of (mM): 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, balanced with 10 HEPES, pH 7.4. To measure [Ca2+]i, we used inverted motorized microscope Leica DMI6000B with a high-speed monochrome CCD-camera HAMAMATSU C9100. For excitation and registration of Fura-2 fluorescence, we used the FU-2 filter set (Leica, Wetzlar, Germany) with excitation filters BP340/30 and BP387/15, beam splitter FT-410, and emission filter BP510/84. Illuminator Leica EL6000 with a high-pressure mercury lamp was used as a source of excitation light.
5
Single-cell calcium imaging of T cells
6
Immunostaining Protocol for Cellular Structures
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For immunostaining, cells were fixed with either 4% PFA for 30 min or 10% TCA for 10 min, permeabilized with 0.2% Triton X-100/PBS for 3 min, and blocked with 1% BSA/PBS for 30 min. The cells were then sequentially incubated for 1 h each with primary and Alexa Fluor–conjugated secondary antibodies diluted in 1% BSA/PBS. The dilutions of the primary antibodies were: Vps35 (1:300), EEA1 (1:300), ezrin (1:300), LAMP2 (1:500), Myc (1:500), TfR (1:300), GalNT2 (1:300), GFP (1:2,000), laminin (1:300), reticulon 4 (1:300), and E-cadherin (1:300). Cysts were finally suspended in PBS and placed on a glass-bottomed dish for microscopy. Fluorescence images were obtained by using FluoView software and a FV1000 confocal laser-scanning microscope (Olympus) equipped with a Plan-Apochromat 63×/1.4 oil-immersion objective lens and an electron-multiplying charge-coupled device camera (C9100; Hamamatsu Photonics). Cropping and level adjustment of the images were performed in Photoshop CS6 software (Adobe).
7
Ratiometric Fluorescence Ca2+ Imaging
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The real-time [Ca2+]i concentration was measured using the ratiometric fluorescence Ca2+ indicator Fura-2 AM (Beyotime), as previous report [42 (link)]. The cells were washed with Hank’s Balanced Salt Solution (HBSS) twice and loaded with 4 μM Fura-2 AM in α-MEM for 30 min in the dark. Fura-2 fluorescence (proportional to [Ca2+]i concentration) was visualized under a microscope (IX71, Olympus) at 340 and 380 nm, emitted from a monochromator (Polychrome V, TILL Photonics GmbH, Grafelfing, Germany). The 510 nm emitted fluorescence was detected using a high-speed cooled CCD camera (C9100, Hamamatsu, Japan) and recorded by Simple-PCI software version 6.60. The [Ca2+]i concentration was calculated as previously described [43 (link)].
8
Live-cell Microscopy Imaging Conditions
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The microscopy measurements were carried out on a Nikon Eclipse Ti microscope (Nikon Instruments Inc., Japan). Images were recorded using a 20×, objective with a numerical aperture of 0.7 (Nikon Instruments Inc., Japan) and a 1000 px × 1000 px EM-CCD camera (C9100, Hamamatsu Photonics K.K., Japan) at temperatures of 19–21 °C. Fluorescence and bright field images were acquired every 8 s for 60–90 min with exposure times below 150 ms.
9
GUV Formation via Electroformation
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GUVs were produced using the electroformation technique. Proteoliposomes were resuspended in 5 mM NaCl and 10 mM Hepes buffer, pH 7.4, to a final 0.5-mg/ml concentration of lipids, and small droplets were spotted on indium tin oxide (ITO)–coated glass slides. Proteoliposomes were partially dehydrated for 2 h in a desiccator under the saturated vapor pressure of KCl solution. The GUV generation chamber was assembled by separating two ITO-coated slides by 0.6 ml of a 300-mM glucose solution and thin rubber spacer. For electroformation, an alternating current electric field was generated by a pulse generator connected to the slides. The field was applied for 3 h across the chamber and incremented continuously from 20 mV to 1.1 V at 12-Hz frequency. Finally, the AC frequency was lowered to 4 Hz at 2 V for 30 min. This lead to detachment of the vesicles from glass slides. GUVs were carefully collected and used immediately for visualization. Images were obtained with a spinning-disk confocal setup (Ultraview; PerkinElmer) that consisted of an inverted microscope (Ti-E Eclipse; Nikon) with a 14-bit electron-multiplying charge-coupled device camera (C9100; Hamamatsu) using Volocity 6.3 software (PerkinElmer).
10
Nanoparticle Effects on Cell Traction Forces
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Cells were seeded at 40% confluence and cell traction forces were determined by traction force microscopy (TFM; Gavara et al., 2006 (link)). Cells were then exposed to Fe2O3 and TiO2 NPs (10 μg/mL) or serum-free medium for 24 h. The samples (n = 7 experiments per group repeated in different days, 7–8 cells/sample) were placed on a microscope (Eclipse Ti, Nikon Instruments, Amsterdam, Netherlands) equipped with a CCD camera (C9100, Hamamatsu Photonics K.K., Hamamatsu, Japan) to measure cell forces. For each traction field, the total force magnitude was computed by integrating the magnitude of the traction field over the projected area of the cell.
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