For time-lapse video microscopy, the neurospheres were dissociated as described in section “2.4 Differentiation and proliferation assay under differentiation conditions.” The NSPCs were plated in a density of 30000 cells per well on poly-L-ornithine (15 μg/ml) coated 24 well plates (Corning, Corning, NY, USA, Cat# 353226) in neurosphere medium. After 2 h, the cells adhered and the medium was supplemented with EGF, FGF2 and heparin in the same concentrations as in the neurosphere culture to ensure proliferation conditions. For the analysis, the produced FnIII Fc recombinants were presented to the cells as coated substrates or as soluble medium additives, both with a concentration of 25 μg/ml. A total of 2 h after seeding the plate was placed in the video microscope system of an Axiovert 200 M provided with an AxioCam HRm and AxioVision-4.8.1 software (Carl Zeiss, Oberkochen, Germany). Additionally, two regulating elements, namely the “Tempcontrol 37-2 digital” and the “CTI-Controller 3700 digital” (PeCon GmbH, Erbach, Germany) ensured stable temperature and pH conditions. Within the system the cells were cultured at 37°C and 5% CO2. For analysis, five spots per domain combination were chosen and a picture was taken every 6 min for 96 h.
Tempcontrol 37 2 digital
Manufactured by Pecon
Sourced in Germany
The Tempcontrol 37-2 digital is a laboratory equipment product designed for temperature control. It features a digital display and electronic controls to maintain a specific temperature within a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (4 mentions)
Axiovision 4, by Zeiss (2 mentions)
Axiocam hrm, by Zeiss (2 mentions)
Cti controller 3700 digital, by Pecon (2 mentions)
Axiocam 506 mono camera, by Zeiss (2 mentions)
Zen software, by Zeiss (2 mentions)
Poly l ornithine, by Corning (1 mentions)
24 well plate, by Corning (1 mentions)
Matlab, by MathWorks (1 mentions)
Glass bottom dish, by Thermo Fisher Scientific (1 mentions)
Observer z1 microscope, by Zeiss (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Lsm 510 meta confocal laser scanning microscope, by Zeiss (1 mentions)
6 protocols using tempcontrol 37 2 digital
1
Time-lapse Microscopy of NSPC Proliferation
2
Microscopic Visualization of Cellular Switching
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Cultures of switcher and control
strains were grown overnight at 30 °C, diluted 100 times into
3 mL of fresh LB, and grown at 30 °C for 2 h. From each culture,
0.7 μL was pipetted into a glass bottom dish (Thermo Scientific,
150682) and covered with a piece of approximately 4 × 4 ×
1 mm 1% LB-agarose pad. A cover glass was placed on top of each pad,
and the dish was covered with a lid to minimize drying. The prepared
cells were observed using a Zeiss Observer Z1 microscope with a 100
×/1.4 oil immersion objective and a Axiocam 506 mono camera (Zeiss).
The switch was induced under the microscope from the beginning of
imaging by maintaining the temperature of the agarose pad at 37 °C
using a Tempcontrol 37–2 digital (PeCon). Images were taken
every 2 min for 6 hours. Multiple positions were imaged in one experiment
using an automated stage and ZEN software (Zeiss). The signals from
GFPmut2 (excitation 460–490 nm; emission 509–550 nm)
and mRFP1 (excitation 540–580 nm; emission 615–675 nm),
together with phase contrast, were recorded.
strains were grown overnight at 30 °C, diluted 100 times into
3 mL of fresh LB, and grown at 30 °C for 2 h. From each culture,
0.7 μL was pipetted into a glass bottom dish (Thermo Scientific,
150682) and covered with a piece of approximately 4 × 4 ×
1 mm 1% LB-agarose pad. A cover glass was placed on top of each pad,
and the dish was covered with a lid to minimize drying. The prepared
cells were observed using a Zeiss Observer Z1 microscope with a 100
×/1.4 oil immersion objective and a Axiocam 506 mono camera (Zeiss).
The switch was induced under the microscope from the beginning of
imaging by maintaining the temperature of the agarose pad at 37 °C
using a Tempcontrol 37–2 digital (PeCon). Images were taken
every 2 min for 6 hours. Multiple positions were imaged in one experiment
using an automated stage and ZEN software (Zeiss). The signals from
GFPmut2 (excitation 460–490 nm; emission 509–550 nm)
and mRFP1 (excitation 540–580 nm; emission 615–675 nm),
together with phase contrast, were recorded.
3
Yeast Cell Imaging and Tracking
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Yeast cultures were grown at 30°C in synthetic complete media with 2% glucose (SC) to OD 0.2-0.6. Cells were pipetted onto 0.8 mm coverglass and covered with a 2% agarose pad made with SC. Before imaging, the cells were allowed to proliferate on the slide for 60 min. Imaging was performed at 30°C using a Zeiss Observer Z1 microscope with a 63X/1.4NA oil immersion objective and Orca-r2 C10600-10B camera (Hamatsu Photonics). Temperature of the agarose pad was held at 30°C using Tempcontrol 37-2 digital from PeCon. Images were taken every 3 min and imaging sessions were 8 h long. Up to 12 positions were imaged using an automated stage and Axiovision software. Definite Focus was used to keep the cells in focus during time-lapse microscopy. NLS-NES-GFP expressing strains were exposed for 15 ms using a Colibri 470 LED module. Clb2- and Cdc6-yeCitrine strains were exposed for 250 ms using Colibri 505 LED module. Spc42-mCherry was imaged using Colibri 540-580 LED module for 750 ms. All Colibri modules were used at 25% power. Image segmentation, cell tracking and quantification of nuclear fluorescence signals was performed using MATLAB (The MathWorks, Inc.) as described in Doncic et al. (2013) (link). For every strain, data are from at least two repeats with different transformants.
4
Visualizing Bacterial Cell State Switching
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Cultures of switcher and control strains were grown overnight at 30°C, diluted 100 times into 3 mL of fresh LB and grown at 30°C for 2 hours. Of each culture, 0.7 mL was pipetted into a glass bottom dish (Thermo Scientific, 150682), covered with a piece of approximately 4 x 4 x 1 mm 1% LB-agarose pad. A cover glass was placed on top of each pad, and the dish was covered with a lid to minimize drying. The prepared cells were observed using a Zeiss Observer Z1 microscope with a 100 × /1.4 oil immersion objective and Axiocam 506 mono camera (Zeiss). The switch was induced under the microscope from the beginning of imaging by maintaining the temperature of the agarose pad at 37°C using a Tempcontrol 37-2 digital (PeCon). Images were taken every two minutes for 6 hours. Multiple positions were imaged in one experiment using an automated stage and ZEN software (Zeiss). The signals from GFPmut2 (excitation 460-490 nm; emission 509-550 nm) and mRFP1 (excitation 540-580 nm; emission 615-675 nm), together with phase contrast, were recorded.
5
Time-lapse Imaging of Adult Neural Stem Cell Proliferation
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For the detailed investigation of adult neural stem cell proliferation behavior, the 24-well plate was placed into the closed system of an Axiovert 200 M equipped with an AxioCam HRm and AxioVision-4.8.1 software (all from Carl Zeiss, Oberkochen, DE). In addition, the two controlling elements ‘Tempcontrol 37-2 digital’ and ‘CTI-Controller 3700 digital’ (PeCon GmbH, Erbach, DE) were used for stable temperature and pH conditions, respectively. Conditions were adjusted 1 h before use. NSPCs were kept in culture medium (DMEM/F12 (#11320-074 Gibco) with 1 × B27 (#17504-044 Gibco), 1 × penicillin/streptomycin (#P4333 Sigma throughout the experiments. The absence of cytokines during the time-lapse studies was intended to uncover selective effects of the culture substrates and to focus on the intrinsic proliferation and differentiation behaviour of aNSPCs, as established in a previous study [8 (link)]. Over a period of 6 days, 8 visual fields per well were documented every 5 min with defined XYZ-coordinates controlled by a moving stage (Märzhäuser, Wetzlar, DE) to ensure a sharp focus over the whole period of time. Thereby, a stack from about 1680 single images can be combined to obtain a time-lapse video with detailed information about the cell behavior in vitro.
6
Visualizing Extracellular Vesicle Internalization
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Labeling of EC-EV and cEV was performed by adding 50 µg/ml CellMask™ orange plasma membrane tracking label for 10 min at 37°C into the supernatant. Free dye was removed from labeled EV using Amicon®Ultra centrifugal columns (10 kDa cutoff) after isolation procedures. Labeled EVs were added to approximately 1 × 106 of HUVECs cell per well in an eight-well culture plate (Ibidi GmbH, Martinsried, Germany). In the case of THP-1, labeled EV were added into poly-d -lysine-coated glass coverslips (Sigma) which were seeded overnight with 8 × 105 undifferentiated THP-1 in six-well plates. Following 2–24 h of incubation, the live cell imagining of internalized of EV was performed using Zeiss LSM 510 META confocal laser scanning microscope (Jena, Germany) on an Axiovert 200 M motorized frame for TICS, STICS, and STICCS analyses. The microscope was coupled to a 30 mW air-cooled argon ion laser emitting at 488 nm under the control of an acousto-optic modulator (~11 µW irradiance at the sample position) for one-photon excitation. To provide a suitable environment for sustaining cells during the imaging steps, the microscope was equipped with an airtight chamber (Tempcontrol 37–2 digital, PeCon, Erbach, Germany) with controlled temperature at 37°C. Cell-free medium-derived EV served as a negative control. Nuclei were stained with Hoechst 33342.
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