MCF10A and MCF10A CDH1-/- cells were seeded at densities of 2.0 × 103 and 4.0 × 103 in three replicates in 96 well E-plates and incubated at 37°C in 5% CO2. The growth rate was monitored in real time at 15 min intervals for 96 h using the xCELLigence platform (Roche). Both cell lines were also seeded at the same densities into 96 well flat clear bottom black plates (Corning) and grown at 37°C in 5% CO2 and imaged every 2 h for 96 h using the IncuCyte 2011A FLR (Essen Bioscience). Confluency was determined using the IncuCyte software Confluence v1.5.
Black clear flat bottom plates
Manufactured by Corning
Sourced in United States
Black/clear flat bottom plates are a type of lab equipment used for various scientific applications. These plates feature a flat bottom design and are available in both black and clear color options. The core function of these plates is to provide a stable and uniform surface for conducting experiments, assays, or other laboratory procedures.
Automatically generated - may contain errors
5 protocols using black clear flat bottom plates
1
Real-Time Cell Growth Monitoring
2
Screening Selenotypus Venom against hNaV1.7
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The crude venom of a female Selenotypus species was screened against hNaV1.7 in SH-SY5Y cells using a Fluorescent Imaging Plate Reader (FLIPRTetra, Molecular Devices, CA, United States) as previously described (Cardoso et al., 2015 (link); Cardoso et al., 2021 (link)). Briefly, SH-SY5Y cells were plated at 40,000 cells per well in 384-well flat clear-bottom black plates (Corning, NY, United States) and cultured at 37°C in a humidified 5% CO2 incubator for 48 h before commencing assays. Cells were loaded with 20 μL per well of Calcium 4 dye (Molecular Devices) reconstituted in assay buffer containing (in mM) 140 NaCl, 11.5 glucose, 5.9 KCl, 1.4 MgCl2, 1.2 NaH2PO4, 5 NaHCO3, 1.8 CaCl2, and 10 HEPES pH 7.4 and incubated for 30 min at 37°C in a humidified 5% CO2 incubator. Fluorescence responses were excited at 470–495 nm and emission recorded at 515–575 nm for 10 s to set the baseline, 600 s after addition of 250, 25 or 2.5 μg/ml venom, and for a further 300 s after addition of 4 μM veratridine and 30 nM scorpion toxin OD1.
3
Enzyme Kinetics of LMVGG Peptide
4
Evaluating SOX5 Influence on Cell Viability
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The effect of SOX5 on cell viability was assessed using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Fitchburg, WI, USA) after transfection with 10 nM control or SOX5 siRNA pools. 1 x 104 cells (fast growing) and 2 x 104 cells (slow growing) cells were seeded per well in 96 well black/clear flat bottom plates (Corning, Corning NY). Viability was measured according to the manufactures instructions 24, 48 and 72 h after transfection. Three biological replicates were performed for each condition.
5
Enzyme Kinetics of LMVGG Peptide
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Assays to determine kcat and Km LMVGG were performed in 300 mM Tris pH 7.5, 150 mM NaCl, 5 mM CaCl2, 5% v/v DMSO, and 10 mM Gly-Gly-Gly-COOH (GGG). The concentration of the Abz-LMVGG(Dnp)-CONH2 peptide substrate ranged from 10 to 200 μM with enzyme concentration of 1 μM. Reactions were conducted in 96-well half area black/clear flat bottom plates (Corning) and initiated with the addition of enzyme. Plates were incubated at 24 °C and monitored for increase in fluorescence (ex=317 nm, em=420 nm) in a Tecan plate reader for 2 hours. Changes in fluorescence were converted to molar velocities using calibration curves of Abz-LMVGG(Dnp)-CONH2 and a 1:1 mixture of free Abz and Dnp. Inner filter quenching effects were corrected using Fcorr = Fobs x antilog[(Aex + Aem)/2], where Fcorr is the corrected fluorescence value, Fobs is the observed fluorescence value, Aex is the absorbance at 317 nm, and Aabs is the absorbance at 420 nm. To determine kcat and Km,LMVGG, initial velocities were fit to the Michaelis-Menten equation in GraphPad Prism.
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