Animals were fasted for 8 h with ad libitum access to autoclaved drinking water. Animals were then orally gavaged with FITC-dextran (3,000–5,000 MW; Sigma) at a dose of 60 mg/100 g body weight. After gavage, animals were housed without food (but given ad libitum access to drinking water) for an additional 4 h and were then euthanized with CO2 per Federal and USC IACUC guidelines. Whole blood was collected from euthanized animals via cardiac puncture. Serum FITC-dextran concentrations were measured using a Synergy 2 fluorescence plate reader (BioTek) with a laser excitation of 485 nm and detection emission wavelength of 528 nm.
Synergy 2 fluorescence plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy 2 is a fluorescence plate reader from Agilent Technologies. It is designed to detect and quantify fluorescent signals in microplates. The device can measure a variety of fluorescent assays, including cell-based and biochemical assays.
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6 protocols using synergy 2 fluorescence plate reader
1
Intestinal Permeability Assay via FITC-Dextran
2
Thapsigargin-Induced Cytoskeletal Changes
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Cells were seeded onto triplicate 24-well plates and grown to confluence. Each cell type was seeded into ten wells of the respective 24-well plate. The corner wells were treated with cell-free endothelial cell media. The WT and constitutive FKBP51 overexpressing PAEC monolayers were treated with either 1 µM thapsigargin in DMSO or the equivalent volume of DMSO without thapsigargin in media for 20 min. The monolayers were paraformaldehyde-fixed, permeabilized, and subjected to cytochemistry using AlexaFluor 568 phalloidin as described above. Nuclear counterstain was also applied as an internal control for cell count. The cell-free corner wells were also treated with the above conditions. Fluorescence at 620-nm and 460-nm wavelengths was recorded in each well simultaneously with a Synergy 2 fluorescence plate reader (BioTek; Winooski, VT) using with Gen5 (BioTek) software. The mean fluorescence from the cell free corner treatments was subtracted from the average fluorescence of treatment at each wavelength measured.
3
Quantifying Fluorescent Protein Levels
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The fluorescence level of the final SGA array strains was evaluated by measuring yEVenus signal in liquid medium. Briefly, the array of colonies were inoculated into liquid leucine dropout SC-MSG medium, and kinetic runs were initiated in a Synergy 2 fluorescence plate reader (Biotek, Winooski, Vermont, United States) for 48 hr, using the following filters: 500/27 (excitation), 528/20 (emission). During the kinetic run, the absorbance (OD600) and yEVenus fluorescence (λex515 nm / λem528 nm) of the growing cultures were monitored simultaneously, with time points taken every 1.5 min. For each time points, the OD600 normalized yEVenus fluorescence (FLOD) was calculated. The fluorescence of a given strain was assessed by calculating the median of the five highest FLOD values.
4
Cell Viability Assay with Alamar Blue
5
Cell Viability Assay Protocol
6
Fluorescence-based Assays for Ion Channel Activity
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K+ channel activity and Em measurements were performed using commercially available FLIPR assays (Molecular Devices, #R8222 and #R8126, respectively), and Fluo-4 assays (Invitrogen, #F36206) for iCa measurements. All three assays were performed following the manufacturer’s instructions. Briefly, for all assays 30,000 cells/well were seeded into dark-walled, clear-bottom 96-well plates (Grenier Bio-One, #655090), and cultured in growth medium overnight. The next day, cells were washed once and incubated at 37 °C with the respective loading dye for 60 min for K+ channel activity assays, and 30 min for Em and Fluo-4 assays. In all assays, fluorescence traces were recorded for 1 min to reach a stable baseline before the addition of any drugs. All plates were analyzed using a BioTek Synergy-2 fluorescence plate reader. Data points were collected and integrated every 7 s. To determine whether an increase in iCa concentrations was due to Ca2+ influx via voltage-gated Ca2+ (CaV) channels, we blocked N- and P/Q-type CaV channels with ω-conotoxin MVIIC (1 μM), and L-type CaV channels with nifedipine (10 μM).
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