All cells were grown at 37°C in medium supplemented with 10% (vol/vol) FBS in a humidified atmosphere containing 5% CO2. MCF-7, SUM159, Hs578T, MDA-MB-231, HeLa, HaCaT and MEF cells were grown in DMEM. HB-2 cells were grown in DMEM supplemented with 10μg/ml insulin (Biological Industries), 5μg/ml Hydrocortisone (Sigma), and 1mM sodium pyruvate. T47D cells were grown in RPMI supplemented with 70μg/ml insulin. Cells were treated with drugs at the following end concentrations and period of time: Noc (0.5 μg/ml) for 18h, EGF (100 ng/ml) for 30 min, Y27632 (10μM) for 20h, peroxovanadate (0.1mM) for 15 min or PD98059 (30μM) for 4h. Actin cytoskeleton was visualized as previously described [7 (link)]. Confocal imaging was performed using Leica TCS STED confocal microscope (Leica microsystems).
Tcs sted confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS STED confocal microscope is a high-resolution imaging system designed for advanced fluorescence microscopy. It utilizes stimulated emission depletion (STED) technology to achieve super-resolution imaging, allowing for the visualization of features beyond the diffraction limit of light. The core function of this microscope is to provide researchers with the ability to capture detailed images of biological samples at nanoscale resolutions.
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4 protocols using tcs sted confocal microscope
1
Cytoskeleton Imaging in Cell Lines
2
Cellular Uptake of PKH67-Labeled EVs
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Initially, 5 × 105 cells were suspended in 500 μL EV-free medium and incubated with PKH67-labeled EVs (2 μg) at 37°C for a specified time, followed by confocal microscopic observation. Cells were frozen, and fluorescence of living cells was observed with Leica TCS-STED confocal microscope (Leica Microsystems, Heidelberg, Germany).
3
FITC-insulin Uptake in MCF-7 Cells
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MCF-7 cells transfected with either Sirt7 siRNA or mock were cultured overnight in chamber slides in MEM medium with a low (0.1%) FBS and at a confluency of 50%. Imaging of fluorescein isothiocyanate-insulin (FITC-insulin) uptake by MCF-7 cells transfected with either Sirt7 siRNA or mock were investigated using a fluorescence microscope (Leica TCS STED Confocal Microscope, Leica Microsystems Ltd., UK). Each experiment was carried out in triplicate.
4
Exosome Trafficking and Intercellular Communication
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Total 5 × 105 cells were re‐suspended in 500 μL exosome‐depleted medium, and incubation was carried out with 2 μg exosomes labelled by PKH‐67 at 37°C. The sample was transferred into ice and underwent 3 washes by means of cold PBS (pH = 7.4). Cells were kept in ice, and fluorescence measurement of living cells was performed with the use of a Leica TCS STED confocal microscope (Leica Microsystems, Wetzlar, Germany).
In Transwell co‐culture experiments, CAF cells were cultured on a Transwell (Thermo Fisher Scientific, Waltham, MA, USA) with 0.4‐mm aperture and transfected with miR‐24‐3p oligo (Biomics Biotechnology Corp, Nantong, China) labelled by 10 nmol/L red fluorescent Cy3 for 24 hours. The cells were then co‐cultured with HT29 cells or SW620 cells on coverslips at the bottom of Transwell. Subsequent to 24 hours, the cells were fixed by means of 4% paraformaldehyde and observed by means of a laser scanning confocal microscopy (LSCM).
In Transwell co‐culture experiments, CAF cells were cultured on a Transwell (Thermo Fisher Scientific, Waltham, MA, USA) with 0.4‐mm aperture and transfected with miR‐24‐3p oligo (Biomics Biotechnology Corp, Nantong, China) labelled by 10 nmol/L red fluorescent Cy3 for 24 hours. The cells were then co‐cultured with HT29 cells or SW620 cells on coverslips at the bottom of Transwell. Subsequent to 24 hours, the cells were fixed by means of 4% paraformaldehyde and observed by means of a laser scanning confocal microscopy (LSCM).
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