VeroE6 cells were transfected with pCC1-4K-SARS-CoV-2-ZsGreen plasmid using Lipofectamine 3000 (Invitrogen) and ZsGreen-expressing viral particles were harvested form culture media, as described by Rihn et al. (2021) (link). VeroE6/TMPRSS2 cells were maintained in DMEM with 200 nM glutamine, 5% FBS and 200 μg/ml Hygromycin-B (Gibco) until seeding at 1.5 × 105 cells/well. After 24 h, cells were transfected with ASOs as described above and infected with SARS-CoV-2/ZsGreen particles at MOI = 0.1 for 24 h. Cells were then detached with 0.05% trypsin (Gibco), and fixed in 4% formaldehyde solution for 30 min. Fluorescence was measured in a TECAN Infinite® 200 PRO plate reader (Tecan Life Sciences, Männedorf, Switzerland) under 480 nm excitation and 530 nm emission in black bottom 96 well plates (Corning).
Black bottom 96 well plate
Manufactured by Corning
Sourced in United States
The Black Bottom 96 Well Plates are a laboratory equipment product designed for use in various scientific experiments and assays. These plates feature a black bottom that helps to minimize background fluorescence and improve signal-to-noise ratio, making them suitable for a range of optical measurements.
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Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Infinite 200 pro plate reader, by Tecan (1 mentions)
Round bottomed ultra low attachment 96 well plates, by Corning (1 mentions)
Synergy neo2 multi mode reader, by Agilent Technologies (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Collagen 3, by Abcam (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Collagen 1, by Abcam (1 mentions)
Alpha actinin, by Abcam (1 mentions)
Alexa fluor 488 anti mouse, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Bio-Techne (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
Safire2 plate reader, by Tecan (1 mentions)
Clear bottom black 96 well plate, by Corning (1 mentions)
Alamarblue, by Corning (1 mentions)
Pherastar, by BMG Labtech (1 mentions)
9 protocols using black bottom 96 well plate
1
SARS-CoV-2 ZsGreen Virus Particle Assay
2
Kaempferol Effects on Skeletal Myoblasts
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The C2C12 mouse skeletal myoblasts were obtained from Pythonbio (Guangzhou, China). The cells were grown in DMEM supplemented with 10% FBS and 1% P/S. At 70–80% confluence, the cells were cultured in DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin for six days to induce differentiation into the myotubes [32 (link)]. All the cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The culture media were replaced every two days.
After differentiation, the cells were seeded at 1 × 104 cells per well into black-bottom 96-well plates (Corning, Kennebunk, ME, USA) overnight for the 2D culture. The cells were seeded at 2000–3000 cells/well into ultra-low attachment 96-well round-bottomed plates (Corning, Kennebunk, ME, USA) and then cultured for three days to generate skeletal muscle spheroids (3D culture) [33 (link)]. The cells were then treated with varying concentrations of kaempferol (50, 25, 12.5, and 6.25 μM) for 24 h in the 2D culture and 48 h in the 3D culture. The treatment concentration of kaempferol was determined based on our previous cell activity experiments. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The testing was subsequently commenced.
After differentiation, the cells were seeded at 1 × 104 cells per well into black-bottom 96-well plates (Corning, Kennebunk, ME, USA) overnight for the 2D culture. The cells were seeded at 2000–3000 cells/well into ultra-low attachment 96-well round-bottomed plates (Corning, Kennebunk, ME, USA) and then cultured for three days to generate skeletal muscle spheroids (3D culture) [33 (link)]. The cells were then treated with varying concentrations of kaempferol (50, 25, 12.5, and 6.25 μM) for 24 h in the 2D culture and 48 h in the 3D culture. The treatment concentration of kaempferol was determined based on our previous cell activity experiments. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The testing was subsequently commenced.
3
GPCR Biosensor FRET Assay in HEK293
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HEK293 cells transfected
with CmorAlstRC variants with Gα sensors were reseeded in black-bottom
96-well plates (Corning) as 75.000 per well. FRET measurements were
performed using a Biotek Synergy Neo2 Multi-Mode Reader, and data
was acquired using Gen5 software (Biotek, Germany). For the measurements,
DMEM was replaced with 90 μL of HBSS. Measurements were performed
using dual monochromators for excitation and emission, and dual PMTs
were used for fluorescence detection. For CFP, excitation and emission
bandwidth were set to 434/20 and 490/20, respectively. FRET was recorded
using the second monochromators, with the bandwidth settings of 434/20
and 540/25 for excitation and emission, respectively. The voltage
gain for both PMTs was set to 100. A xenon lamp was set to low-energy
mode, read height was set to 4.5 mm, and read speed was set to normal.
Data points for each well were generated with 90 s intervals. Four
data points per well were measured for the baseline. After the baseline
measurement, cells were stimulated manually with the ligands using
a multichannel pipette, and 5 more data points were acquired per well.
For each concentration, 8 wells were used and the results were averaged.
FRET was calculated as the ratio of FRET and CFP values for each
data point of each well. FRET changes due to different concentrations
of the ligand (% ΔFRET) were calculated using the following
formula
with CmorAlstRC variants with Gα sensors were reseeded in black-bottom
96-well plates (Corning) as 75.000 per well. FRET measurements were
performed using a Biotek Synergy Neo2 Multi-Mode Reader, and data
was acquired using Gen5 software (Biotek, Germany). For the measurements,
DMEM was replaced with 90 μL of HBSS. Measurements were performed
using dual monochromators for excitation and emission, and dual PMTs
were used for fluorescence detection. For CFP, excitation and emission
bandwidth were set to 434/20 and 490/20, respectively. FRET was recorded
using the second monochromators, with the bandwidth settings of 434/20
and 540/25 for excitation and emission, respectively. The voltage
gain for both PMTs was set to 100. A xenon lamp was set to low-energy
mode, read height was set to 4.5 mm, and read speed was set to normal.
Data points for each well were generated with 90 s intervals. Four
data points per well were measured for the baseline. After the baseline
measurement, cells were stimulated manually with the ligands using
a multichannel pipette, and 5 more data points were acquired per well.
For each concentration, 8 wells were used and the results were averaged.
FRET was calculated as the ratio of FRET and CFP values for each
data point of each well. FRET changes due to different concentrations
of the ligand (% ΔFRET) were calculated using the following
formula
4
Immunofluorescence Quantification of ECM Proteins
5
Immunocytochemistry of hiPSC-Derived Cardiomyocytes
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For immunostaining, hiPSC-CMs were plated on thin black bottom 96 well plates (Corning). Cells were washed using PBS followed by fixing using 5% PFA (Sigma-Aldrich) for 15 min followed by 2x PBS washes. Fixed cells were then washed for 15 min in 0.1% Triton X-100 (Sigma-Aldrich, Gillingham, UK) in PBS followed by 3x further washes in PBS. Fixed cells were then blocked in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS for 1 h followed by a further 5 min PBS wash. Primary antibody was added to the plates, diluted in 4% Goat Serum in PBS (Alpha Actinin 1:800, Abcam) and left overnight at 4 °C on a rocking plate. The following day fixed cells were washed with 0.1% Tween (Sigma-Aldrich, Gillingham, UK) in PBS (3 × 10 min) followed by secondary antibody staining [Anti-Mouse Alexa Fluor® 488 (Abcam, Cambridge, UK) diluted 1:500 in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS] for 1 h at room temperature. The fixed cells were then washed 3 times with 0.1% Tween-PBS (Sigma-Aldrich, Gillingham, UK) followed by incubation in PBS supplemented with DAPI (Tocris, Abingdon, UK) at 1:500 for 10 min. A further wash was performed using PBS after which the fixed cells were submerged in PBS and the plate wrapped in foil prior to imaging using the EVOS M5000 imaging system.
6
Metabolic Assessment of Cell Cultures
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Alamar Blue (Invitrogen, Waltham, MA, USA) was used to evaluate the overall metabolic status of ECB-NGF devices or NGC0211 cells. Briefly, NGC-0211 cells (1 × 104 cells/well/100 µL) were seeded in clear bottom 96-well black plates (Corning, New York, NY, USA) for 24 h and exposed to control media or MCMs for 3 and 24 h, respectively. In the meantime, 10 µL of 10× Alamar Blue was added to every well during the last 1 h of treatment incubation. Fluorescence was then read in a spectrophotometer (Safire II Plate reader, Tecan, Männedorf, Switzerland; 5 nm bandpass; top read) at 560/590 nm. Similarly, post 7 weeks of experimentation with ECB-NGF devices, 50 µL of 10× Alamar Blue was added in 500 µL DMEM/F12 medium containing 5% FBS and incubated for 1 h. From each well, 100 µL was drawn in triplicate, plated in black bottom 96-well plate (Corning, New York, NY, USA) and the fluorescence was read as mentioned above.
7
Fluorescence-based Rap1B Nucleotide Exchange
8
Characterizing 3D Cell Viability and DNA
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Cell viability was visualized with Live-Dead staining solution (containing 8 µg/mL fluorescein diacetate and 20 µg/mL propidium iodide-both Sigma Aldrich, St. Louis, MO, USA) after 7 days of 3D culture, as previously described [23 (link)]. Furthermore, the amount of dsDNA within the scaffolds was quantified with the Invitrogen™ Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) after 7 and 28 days. Scaffolds were harvested and prepared as described by Wiggenhauser et al. [19 (link)]. For the fluorometric measurement of the samples, Tris-EDTA-Buffer was added in a ratio of 1:3, and 100 µL of the solution were diluted 1:1 with Quant IT PicoGreen dsDNA reagent. Fluorescence was measured in a black bottom 96-well plate (Corning, NY, USA) at 504 nm extinction and 550 nm emission wavelength with a plate reader (Tecan SAFIRE II, Tecan Group, Maennedorf, Switzerland). The volume of DNA was calculated in ng/mL against a dsDNA standard curve that was prepared of a serially diluted Lambda DNA standard.
9
Assessing Caco-2 Monolayer Permeability
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Permeation to uncharged molecules was assessed via unidirectional 4 kDa FITC-dextran (Sigma, Burlington, MA, USA) flux. After incubation of filter-grown Caco-2 monolayers with 13-HPODE, monolayers were washed with PBS and supplied with fresh phenol red free-serum free medium. Thereafter, 1 μg/mL FITC-dextran was added to the top chamber in which 100 μL aliquots were removed from the bottom well and added to a black-bottom 96-well plate (Corning, New York, NY, USA) in triplicates at indicated time points. An equal amount of medium was returned to the bottom wells to preserve volume. Fluorescence was measured in a PerkinElmer Envision 2104 Multilabel Plate Reader at an excitation wavelength of 490 nm and emission wavelength of 520 nm. For consistency, fluorescent flux values obtained 30 min after addition of dextran to top well were used.
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