The cytosolic Ca2+ response was evaluated using the fluorescent Ca2+ indicator Fura-2/AM (Thermo Fischer Scientific). In brief, cells were grown on 24-mm coverslips and incubated at 37 °C for 30 min in 1 mM Ca2+ in Krebs-Ringer buffer (KRB: 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM K2HPO4, 5.5 mM glucose, 20 mM HEPES) supplemented with 2.5 mM Fura-2/AM, 0.02% Pluronic F-68 (Sigma-Aldrich), and 0.1 mM sulfinpyrazone (Sigma-Aldrich). Cells were then washed and supplied with 1 mM Ca2+/KRB. Next, cells were placed in an open Leyden chamber on a 37 °C thermostated stage and exposed to 340/380 nm wavelength light using the Olympus xcellence (Olympus) multiple wavelength high-resolution fluorescence microscopy system equipped with an Hamamatsu ORCA ER CCD camera (Hamamatsu Photonics) and a Upl FLN 40× oil objective (Olympus) to determine the cytosolic Ca2+ response. The photo-activation of aluminium phthalocyanine chloride was obtained using an excitation filter ET576/25 (Semrock), with 500 ms of excitation every cycle. Cytosolic Ca2+ concentration was calculated as previously described19 (link),20 (link).
Xcellence
Manufactured by Olympus
Sourced in Hungary, Germany, Japan
The Xcellence is a high-performance lab equipment product. It is designed for precise and efficient laboratory tasks. The core function of the Xcellence is to provide reliable and consistent performance in a laboratory setting.
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Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (3 mentions)
Fura 2 am, by Merck Group (3 mentions)
Pluronic f68, by Merck Group (3 mentions)
Sulfinpyrazone, by Merck Group (3 mentions)
Et576 25, by IDEX Corporation (3 mentions)
Orca r2 cooled digital camera, by Hamamatsu Photonics (3 mentions)
Hamamatsu orca er ccd camera, by Hamamatsu Photonics (2 mentions)
Uplfln 40 oil objective, by Olympus (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ix81 inverted microscope, by Olympus (2 mentions)
Live dead assay, by Thermo Fisher Scientific (2 mentions)
Ethidium homodimer, by Thermo Fisher Scientific (2 mentions)
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Inubg2e onics, by Tokai Hit (1 mentions)
C9100 13 em ccd camera, by Hamamatsu Photonics (1 mentions)
Spss v19, by IBM (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
18 protocols using xcellence
1
Cytosolic Calcium Response Evaluation
2
Cytosolic Calcium Response Evaluation
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The cytosolic Ca2+ response was evaluated using the fluorescent Ca2+ indicator Fura-2/AM (Thermo Fischer Scientific). In brief, cells were grown on 24-mm coverslips and incubated at 37 °C for 30 min in 1 mM Ca2+ in Krebs-Ringer buffer (KRB: 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM K2HPO4, 5.5 mM glucose, 20 mM HEPES) supplemented with 2.5 mM Fura-2/AM, 0.02% Pluronic F-68 (Sigma-Aldrich), and 0.1 mM sulfinpyrazone (Sigma-Aldrich). Cells were then washed and supplied with 1 mM Ca2+/KRB. Next, cells were placed in an open Leyden chamber on a 37 °C thermostated stage and exposed to 340/380 nm wavelength light using the Olympus xcellence (Olympus) multiple wavelength high-resolution fluorescence microscopy system equipped with an Hamamatsu ORCA ER CCD camera (Hamamatsu Photonics) and a Upl FLN 40× oil objective (Olympus) to determine the cytosolic Ca2+ response. The photo-activation of aluminium phthalocyanine chloride was obtained using an excitation filter ET576/25 (Semrock), with 500 ms of excitation every cycle. Cytosolic Ca2+ concentration was calculated as previously described19 (link),20 (link).
3
Cytosolic Ca2+ Response Measurement
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The cytosolic Ca2+ response was evaluated using the fluorescent Ca2+ indicator Fura-2/AM (Life Technologies, Invitrogen). The MEFs were grown on 24-mm coverslips and incubated at 37 °C for 30 minutes in 1 mM Ca2+/KRB supplemented with 2.5 mM Fura-2/AM, 0.02% Pluronic F-68 (Sigma-Aldrich), and 0.1 mM sulfinpyrazone (Sigma-Aldrich). Then, the cells were washed and supplied with 1 mM Ca2+/KRB. Next, the cells were placed in an open Leyden chamber on a 37 °C thermostated stage and exposed to 340/380 wavelength light using the Olympus xcellence (Olympus) multiple wavelength high-resolution fluorescence microscopy system equipped with an Hamamatsu ORCA ER CCD camera (Hamamatsu Photonics) and a Upl FLN 40x oil objective (Olympus) to determine the cytosolic Ca2+ response. The photo-activation of aluminum phthalocyanine chloride was obtained using an excitation filter ET576/25 (Semrock), with 500 milliseconds of excitation every FRET ratio cycle. The collected fluorescence data are expressed as emission ratios.
4
Immunofluorescent Localization of BCAT2
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Cells grown in 24-well plates with glass coverslips on the bottom were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 3 min. Samples then were incubated with primary antibody against BCAT2 (Abcam, Cambridge, UK) for 1 h at 37 °C, then rinsed with 1% BSA/PBS and incubated with secondary donkey anti-rabbit IgG H&L antibody conjugated with Alexa Fluor 488 (Life technologies, Carlsbad, CA, USA) for 30 min. Coverslips were attached with a Vectashield Mounting Medium with DAPI (Vector Laboratories, CA, USA). The analysis was performed with an inverted fluorescence microscope Olympus IX81 (Olympus Europa holding Gmbh, Hamburg, Germany) equipped with an Orca-R2 cooled digital camera (Hamamatsu Photonics K.K., Japan), the fluorescence excitation system MT10 (Olympus Life Science Europa Gmbh, Hamburg, Germany), and the fluorescence imaging system XCELLENCE (Olympus Soft Imaging Solutions Gmbh, München, Germany).
5
Time-lapse imaging of LSCC cell motility
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Time-lapse imaging of LSCC cell motility and TT formation in the culture medium was performed at 37°C in a humidified atmosphere of 5% CO2 using an incubation system INUBG2E-ONICS (Tokai Hit, Shizuoka-ken, Japan) with an incubator, mounted on the stage of the motorized Olympus IX81 microscope (Olympus Europe holding Gmbh, Hamburg, Germany) equipped with UPLFLN 4x/0.13, UPLFLN 10x/0.3, UPlanSApo 20x/0.85 OI, or PlanApo N 60x/1.42 OI lens, the Orca-R2 cooled digital camera (Hamamatsu Photonics K.K., Japan) and the fluorescence imaging system XCELLENCE (Olympus Soft Imaging Solutions GmbH, München, Germany). Differential interference contrast (DIC) or phase-contrast images were taken in addition to time-lapse imaging.
6
Immunoelectron Labeling of CD31 in Tumor Tissue
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Tumor specimens were fixed in formalin overnight, embedded in paraffin, and cut into 4 mm thick series sections. For immunoelectron labeling, the tissue sections were performed by incubating with CD31 antibodies overnight at 4°C. The sections were then reacted with appropriate secondary antibodies. The entire section was scanned by a scanning system that consists of fluorescence microscope (Xcellence, Olympus, Tokyo, Japan), Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, Maryland), and SPSS V19.0.
7
Quantitative Analysis of Brown Adipocyte Lipid Accumulation
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Non-differentiated brown adipocytes were seeded overnight at a density of 1.5×104 per well on 35 mm glass bottom culture dishes (Glass No. 0, MatTek Corporation) and differentiated into mature brown adipocytes. At days 1, 3, 6 and 9 of differentiation the brown adipocytes were washed twice with PBS and fixed with 4% paraformaldehyde at 4°C overnight. Thereafter fixed brown adipocytes were washed three times with PBS and stained with 3.7 mM freshly prepared Oil Red O (ORO) solution at room temperature. After a 20 min ORO incubation, brown adipocytes were washed three times with PBS and examined under an Olympus IX81 inverted microscope. Microscopic images were post-processed using the Olympus-specific software Xcellence (Olympus).
8
Cardiomyocyte Ca2+ Transient Analysis
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Cardiomyocytes from each adenovirally cotransduced group were split in two (DMSO control and mavacamten treatment). After 15 min incubation at 37°C, cardiomyocytes were imaged on an Olympus IX81 inverted microscope (Olympus, Japan) with a C-9100-13 EMCCD camera (Hamamatsu, Japan). Videos of 0.5-Hz electrically paced cardiomyocytes at 37°C were acquired at 25 frames/s (560/25 nm excitation, 620/60 nm emission with a 565 nm dichroic mirror). Raw image data were extracted using xcellence (Olympus), analyzed in Excel (Microsoft) and Ca2+ transient parameters extracted; all baseline variation of Ca2+ transients derived from differential expression of RGECO or driven by altered basal sarcomere lengths (via myofilament Ca2+ buffering) were normalized by calculating ΔF/F for each transient (17 (link)).
9
Fluorescent Labeling of HKs and HCECs
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The red fluorescent protein (RFP) and the green fluorescent protein (GFP) labelling kits were purchased from Shanghai Zhongqiao Xinzhou Co. (Shanghai, China). According to the manufacturer’s instructions, the fluorescently labeled cells continuously express fluorescent protein after proliferation.
Fluorescently labeled HKs (red) and HCECs (green) were used to observe the proliferation of these cells on the electrospun scaffolds and the effect of the aligned and non-aligned topological structure on cells was observed by confocal microscopy (Xcellence, Olympus, Hong Kong, China). The method for the cell seeding is described inSection 2.6 . After 1, 2, 3, and 4 weeks of culture, confocal microscopy was used to observe the HKs and HCECs on the hybrid scaffolds by adjusting the focal plane and excitation wavelength.
Fluorescently labeled HKs (red) and HCECs (green) were used to observe the proliferation of these cells on the electrospun scaffolds and the effect of the aligned and non-aligned topological structure on cells was observed by confocal microscopy (Xcellence, Olympus, Hong Kong, China). The method for the cell seeding is described in
10
Measurement of Intracellular Calcium
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Ca2+-sensitive fluorescent dye FURA 2AM (5 µM) was used for the measurement of intracellular Ca2+ concentration [Ca2+]i as described earlier.5 (link) Changes in [Ca2+]i were measured using an imaging system (Xcellence; Olympus, Budapest, Hungary). Four to 5 small areas (region of interests [ROIs]) of 5 to 10 cells in each intact duct were excited with light at 340 nm and 380 nm, and the 380 / 340 fluorescence emission ratio were measured at 510 nm. One [Ca2+]i measurement was obtained per second.
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