In order to assess the endothelial cell phenotype of tube formation in the iChEC-1 line, Matrigel (Corning Incorporated, Life Sciences, MA, USA, 354234) was thawed at 4C and diluted in EC medium to a concentration of 10 mg/mL. A volume of 290 μL was dispensed with cooled pipet tip to a 24-well flat-bottom tissue culture plate (Nunc Cat. No. 142475, Thermo Scientific). The plate was subsequently incubated for 1 hr at 37°C, followed by addition of ChECs (trypsinized at approximately 80% confluency). A total of 300 μL of the cell suspension was added to each well, at densities ranging from 3.7×l04 to 3×l05. The plate was incubated for 18 to 24 hours at 37°C and 5% CO2. Following incubation, medium was carefully removed and the wells were washed twice with HBSS. Cells were labeled by adding 300 μL of Calcein AM dye per well (Corning, 8 μg/mL in HBSS) and incubating for 1 h at 37°C, 5% CO2, followed by photomicrography on an inverted fluorescence microscope (1X81, Olympus, Tokyo, Japan).
Calcein am dye
Manufactured by Corning
Sourced in Japan, Germany
Calcein AM dye is a fluorescent cell-permeable indicator used to detect viable cells. It is a non-fluorescent compound that becomes fluorescent upon intracellular esterase activity, indicating the presence of living cells.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using calcein am dye
1
Endothelial Cell Tube Formation Assay
2
Assessing PA's Effects on Neurons
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The effect of PA on neurons alone or against CDDP-treated DRG cells
was studied in terms of axon length changes. 200 μL of medium
containing 1000 DRG cells was seeded on each PDL/laminin-coated 96-well
glass-bottom plate. After 24 h of culture, the cells were treated
with CDDP, PA, and a combination for another 24 h. PA was administered
across a range of concentrations, including 0.002, 0.02, 0.09, 0.9,
1.8, 4.5, and 9 mM. CDDP was used at concentrations of 1, 2, 5, 10,
20, and 50 μM in a separate set of experiments. After 24 h of
drug treatment, the cells were stained with 5 μM calcein-AM
dye (Corning)-containing media, followed by live-cell imaging by a
fluorescence microscope (Leica DMi8, Germany). ImageJ software (Java
1.8.0_345 (64-bit)) was used to estimate the length of the axons.
At least 60 axons were selected from triplicate samples to calculate
the average axon length. To evaluate neuronal survival, the cells
were cultured for 7 days to get axon confluence in wells. Later, the
DRG cells were subjected to culture under CDDP, PA, and CDDP–PA
for 7 days. The cell survivability was evaluated by counting the live
cells and determining the percentage of cells relative to the control
group, which was considered 100%. Three frames of each well of triplicate
samples were used to estimate the average numbers.
was studied in terms of axon length changes. 200 μL of medium
containing 1000 DRG cells was seeded on each PDL/laminin-coated 96-well
glass-bottom plate. After 24 h of culture, the cells were treated
with CDDP, PA, and a combination for another 24 h. PA was administered
across a range of concentrations, including 0.002, 0.02, 0.09, 0.9,
1.8, 4.5, and 9 mM. CDDP was used at concentrations of 1, 2, 5, 10,
20, and 50 μM in a separate set of experiments. After 24 h of
drug treatment, the cells were stained with 5 μM calcein-AM
dye (Corning)-containing media, followed by live-cell imaging by a
fluorescence microscope (Leica DMi8, Germany). ImageJ software (Java
1.8.0_345 (64-bit)) was used to estimate the length of the axons.
At least 60 axons were selected from triplicate samples to calculate
the average axon length. To evaluate neuronal survival, the cells
were cultured for 7 days to get axon confluence in wells. Later, the
DRG cells were subjected to culture under CDDP, PA, and CDDP–PA
for 7 days. The cell survivability was evaluated by counting the live
cells and determining the percentage of cells relative to the control
group, which was considered 100%. Three frames of each well of triplicate
samples were used to estimate the average numbers.
3
In Vitro Angiogenesis Assay Using iHUVECs
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