BAM‐transduced fibroblasts were cultured for 6 weeks before being characterized for intracellular calcium signals using Fluo‐4, AM (Invitrogen, F14201) according to the manufacturer's protocol. Briefly, Fluo‐4 AM (2µm ) was diluted in phenol‐red free conditioned media, and cells were incubated with the conditioned media plus the calcium indicator for 1 h in a 37 °C humidified CO2 incubator. Just before imaging, cells were washed with 37 °C pre‐warmed Hank's balanced salt solution (HBSS) and incubated with 37 °C pre‐warmed phenol red‐free media. Green fluorescence intensity of Fluo4 was recorded using a fluorescence microscope (BZ 7000, Keyence, Japan) in 7.4 frames per second using a 10X objective on a 37 °C heating plate. Entire fields of view (1360‐by‐1024 pixels) were segmented as 20‐by‐20 pixels, and every frame of the movies was analyzed and plotted as a time versus fluorescence intensity graph using MATLAB (MathWorks, USA). To quantify reprogramming efficiency, cells with neuron morphology and calcium sparking were counted and normalized with the number of initially seeded cells.
Bz7000
Manufactured by Keyence
Sourced in Japan
The BZ7000 is a digital microscope that captures high-quality images and videos. It features a CMOS camera with up to 4K resolution and advanced image processing capabilities. The BZ7000 is designed for detailed observation and analysis of samples in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Streptavidin alexa 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Biotinylated griffonia, by Vector Laboratories (1 mentions)
Cm1860 cryostat, by Leica (1 mentions)
4 protocols using bz7000
1
Characterizing Calcium Signaling in BAM-Transduced Fibroblasts
2
Immunofluorescence Staining of Endothelial Cells
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At a chosen time, Langendorff-perfused hearts were cut and incubated in 4% ice-cold PFA for 1 hour, followed by overnight incubation in 30% sucrose at 4°C. The hearts were then frozen with OCT compound using liquid nitrogen, from which 7.5 μm transverse cryosections were cut. The sections were blocked with 10% goat serum for 60 minutes and then incubated with 1/100 Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I Isolectin B4 antibody (Vector laboratories, 0.5mg/ml) in 5% goat serum for 60 minutes. Isolectin-B4 is a marker for endothelial cells [20 (link)]. After washing, sections were incubated with 1/400 dilution of Streptavidin—Alexa 594 (Invitrogen, UK, 2 mg/ml, Catalogue number s32356), together with 1/1000 dilution of DAPI (Roche, Catalogue: 10236276001) for nuclear counterstaining, for a further 60 minutes. The sections were observed using fluorescent microscopy (BZ7000, Keyence) with DAPI-B, GFP and TxRed filters (Keyence).
3
Intracranial Aneurysm Morphometry
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Bifurcations of the right olfactory artery and the anterior cerebral artery were stripped and frozen in OCT compound. Samples were sliced 5‐μm thick with a Cryostat CM1860 (Leica, Wetzlar, Germany) and stained with Elastica van Gieson. Four parameters of the IAs were measured: aneurysm size, (maximum height plus maximum width of the lumen) divided by 2; size of the internal elastic laminar disruption, (end‐to‐end dimension of internal elastic laminar plus length of perpendicular line from tip of the aneurysm) divided by 2; wall thickness ratio, minimum width of aneurysmal wall divided by average thickness of normal arterial wall; and lumen area of aneurysms, calculated by the BZ‐7000 (Keyence, Osaka, Japan).
4
Particle Shape Influence on Cellular Uptake
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The effect of the particle shape on cellular uptake
was determined by culturing RAW 264.7, in the presence of Nile Red-loaded
spherical or rod-shaped particles and counting the number of uptakes.
RAW 264.7 was seeded at a cell concentration of 2.0 × 104 cells/cm2 and incubated at 37 °C for 24 h
in FBS-containing DMEM. After removing the medium and washing with
sterilized PBS, 900 μL of FBS-containing DMEM was added. PBS
(100 μL) containing the sample particles was then added at a
concentration of 1 mg/mL (final particle concentration of 100 μg/mL)
and incubated at 37 °C for 24 h. The medium was removed, 10%
(v/v) formalin was added, and the mixture was left to stand for 10
min for cell fixation. The number of particles taken up by RAW 264.7
cells was counted using a fluorescence microscope (BZ7000, Keyence,
Osaka, Japan).
was determined by culturing RAW 264.7, in the presence of Nile Red-loaded
spherical or rod-shaped particles and counting the number of uptakes.
RAW 264.7 was seeded at a cell concentration of 2.0 × 104 cells/cm2 and incubated at 37 °C for 24 h
in FBS-containing DMEM. After removing the medium and washing with
sterilized PBS, 900 μL of FBS-containing DMEM was added. PBS
(100 μL) containing the sample particles was then added at a
concentration of 1 mg/mL (final particle concentration of 100 μg/mL)
and incubated at 37 °C for 24 h. The medium was removed, 10%
(v/v) formalin was added, and the mixture was left to stand for 10
min for cell fixation. The number of particles taken up by RAW 264.7
cells was counted using a fluorescence microscope (BZ7000, Keyence,
Osaka, Japan).
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