Cell viability and proliferation assay were performed as described in our previous study [16 (link)]. Briefly, AML cells were seeded into 96-well plates (3 × 103 per well) and treated with indicated concentrations of compounds for 72 h. Cell viability was then determined by MTS assay (Promega; Madison, WI), and optical density was measured at 490 nm using a CytationTM 5 Imaging Multi-Mode reader (BioTek, Winooski, VT). Cells were counted for proliferation determination. Cell viability and cell proliferation were uniforms as a percentage versus control (vehicle set at 100%).
Cytation 5 imaging multi mode reader
Manufactured by Agilent Technologies
Sourced in United States
The Cytation 5 Imaging Multi-Mode Reader is a high-performance, automated cell imaging and analysis system. It combines advanced cell imaging capabilities with multi-mode detection in a single compact instrument. The Cytation 5 is designed to capture, analyze, and quantify cellular and subcellular features.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Mts assay, by Promega (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Luciferase substrate, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Preadipocyte medium, by ZenBio (1 mentions)
Adipocyte medium, by ZenBio (1 mentions)
Dapi solution, by Solarbio (1 mentions)
3t3 l1 preadipocytes, by ZenBio (1 mentions)
Prism 9, by GraphPad (1 mentions)
Verapamil, by MP Biomedicals (1 mentions)
Mitoxantrone, by Santa Cruz Biotechnology (1 mentions)
Ko143, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Biosynth (1 mentions)
Cyanine 5 azide, by Lumiprobe (1 mentions)
Lev simplyrna tissue kit, by Promega (1 mentions)
36 protocols using cytation 5 imaging multi mode reader
1
Cell Viability and Proliferation Assay
2
Quantifying Oxidative Stress in Fungal Strains
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Reactive oxygen species levels were quantified using the probe CM-H2DCFDA (Molecular probes, Invitrogen, Eugene, OR, USA). Both fungal strains were cultivated in minimum media for 24 h in a 96-well plate. Each test condition (different time of exposure to H2O2) was repeated in octuplicate. H2O2 was added to growth media to a final concentration of 5 mM before incubation with a 0.25 mM CM-H2DCFDA probe for 30 min at 30 °C, and then washed twice with Phosphate-Buffered Saline pH 7.4 (PBS). Fluorescence (485/535 nm) was measured immediately using CytationTM 5 Imaging Multi-mode Reader (BioTek, Winooski, VT, USA).
3
Tango Assay for GPR132 and ARRB2 Interaction
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Tango assay was performed as described in our previous study [21 (link)]. CHO cells cultured in a 6-cm dish were co-transfected with GPR132-Tango, ARRB2-TEV, and TRE-Luc with a ratio of 2:1:1 by using polyethyleneimine (PEI). Transfected cells were then seeded into a 96-well plate at a density of 2 × 104 cells per well. After 24 h, the medium was refreshed, and cells were incubated with compounds at indicated concentrations for 18 h. Subsequently, cells were lysed with Passive Lysis buffer (Promega, Madison, WI, USA) for 15 min at room temperature in a shaker at 70 rpm. The cell lysate was transferred to white 96-well plates, and the bioluminescence was determined immediately after the addition of luciferase substrate (Promega, Madison, WI, USA) with a CytationTM 5 Imaging Multi-Mode reader (BioTek, Winooski, VT).
4
Skin Biopsy Culture and Compound Treatment
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Human skin experiments utilized skin obtained from discarded skin from panniculectomies and other surgical procedures. Patient consent for experiments was not required because deidentified, leftover surgical human tissue is considered to be discarded material by our institution, and thus the studies were exempt. Small, 8 mm punch biopsies were excised and placed in a 24-well Millicell cell culture insert in a well of a 24-well plate containing 0.5 ml of culture medium (DMEM [Hyclone, Logan, UT] containing 10% fetal bovine serum [Hyclone] and a combination of penicillin and streptomycin [Life Technologies, Grand Island, NY]) (Choi et al., 2020 (link); Kemp et al., 2019 (link)). For addition of compounds to the culture medium, 1 μl of DMSO or a 10 mM stock of SP or Can (Sigma-Aldrich) were added to the medium (to obtain a final concentration of 20 μM). For topical treatments, 25 μl of a 120 mM (5%) SP or Can solution (or DMSO vehicle) was pipetted onto the epidermis of the biopsy. Treatments were repeated 24 hours later, and then the biopsies were snap frozen in liquid nitrogen 48 hours after the first treatment. Additional biopsies were fixed in formalin and then embedded in paraffin, sectioned, and stained with H&E by AML Laboratories (St. Augustine, FL). Slides were visualized using a Cytation 5 Imaging Multi-Mode Reader (BioTek, Winooski, VT).
5
Differentiation of 3T3-L1 Preadipocytes
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3T3-L1 preadipocytes were obtained from ZenBio (North Carolina, USA) and cultured in preadipocyte medium (ZenBio) at 37 °C under 5% CO2 in a humidified incubator. To induce differentiation into mature adipocytes, 3T3-L1 cells were cultured to 100% confluence (Day 0). Two days later (Day 2), the culture medium was replaced with differentiation medium (ZenBio) and cells were cultured for a further three days. Subsequently, the cells were cultured in adipocyte medium (ZenBio), which was refreshed daily until Day 11 when full differentiation into mature adipocytes was achieved. Lipid droplets were stained with oil red O (ORO) and LipidSpotTM 610 as previously described 23 . In brief, for ORO staining, differentiated 3T3-L1 cells were fixed with 4% PFA, rinsed twice with 60% isopropanol and then stained with ORO (Sigma, 0.6% in 60% isopropanol) for 30 min at room temperature. Stained samples were then rinsed twice with 60% isopropanol and then with PBS. For fluorescence staining, cells were fixed with 4% paraformaldehyde (PFA) and then permeabilized with 0.1% Triton X-100 for 10 min. The cells were incubated first with LipidSpotTM 610 (1:1,000, Biotium) for 10 min followed by DAPI solution (Solarbio) for 5 min at room temperature, avoiding light exposure. Images were captured with the Cytation 5 Imaging Multimode Reader (Biotek).
6
Crowder-Modulated DNA Strand Displacement
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Annealing of the FRET pair duplex oligonucleotides (Bmut-CoBmut, 1000 nM in a 100 µL) was performed by exposing the mixture to 90 °C for 10 min and cooling down at room temperature for 2 h, followed by chilling at +4 °C for 1 h. Once the duplex Bmut-CoBmut was formed, it exhibited FRET upon excitation. Next, the crowders, peg 45%, deg 45%, and pvp 10% (concentrations given as w/w) were introduced for a minimum time of 15 min. Immediately after the addition of the displacement strand (DS1, DS2, DS3, or DSCtrl, in a five-fold molar excess), the Cy5 fluorescence emission over time was measured on a BioTek Cytation 5 Imaging Multi-Mode reader for a minimum of 30 min. To monitor FRET, we excited the donor Cy3 dye at 535 nm and followed the fluorescence emission of the acceptor Cy5 at 675 nm. We measured the kinetics in a 37 °C temperature-controlled instrument. As the displacement reaction proceeds, the FRET pair dissociates, resulting in a decrease in the FRET efficiency between Cy3 and Cy5, and decreased Cy5 fluorescence emission.
7
Real-time Monitoring of Ca-DPA Release
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Ca-DPA (Calcium dipicolinic acid) release was monitored in real time using a terbium fluorescence assay55 (link),34 (link). An opaque 96-well plate was seeded with 125 µl/well of the germination solution supplemented with 800 µM of TbCl3. Heat (65 °C)-activated spores were sedimented for 1 min at 14,000 × g, and resuspended in an equal volume of water. A 5 µl sample of a spore suspension (with an OD600 of 30) was added to each well, and the Ca-DPA release was monitored using a Molecular Devices Cytation™ 5 Imaging Multi-Mode Reader (BioTek, USA) (excitation, 270 nm; emission, 545 nm; cutoff, 420 nm)55 (link).
8
Histopathology and Lipid Detection
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H&E staining was performed on 4 μm sections prepared from tissues fixed with 4% PFA and embedded in paraffin. Neutral lipids were detected in ORO staining of 7 μm sections prepared from tissues embedded in optimal cutting temperature (OCT) compound. After fixation with 4% PFA, sections were stained with 0.3% ORO following standard procedures. Images were captured with the Cytation 5 Imaging Multimode Reader (Biotek).
9
pH-Dependent Amyloid-Beta Fluorescence
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The cell culture medium was supplemented with dilute HCl and NaOH solutions to obtain different solutions of pH ranging from 1.0 to 9.0 for the assay. Lyophilized powder of Aβ conjugated with pHrodo (RODO–AβpH) and Aβ conjugated with Protonex Green® (PTXG–AβpH) was dissolved in cell culture medium to make stock solutions and kept at 37 °C for 24 hours to pre-aggregate the peptide conjugates. From the stock solutions, different dilutions for each pH condition were prepared at concentrations of 0.5, 1.0, 2.0, and 5.0 μM in a 96-well plate (100 μL per well). Fluorescence intensity of each well containing AβpH was obtained on a Cytation™ 5 imaging multi-mode reader (BioTek Instruments) at 443/505 nm excitation/emission wavelengths. The fluorescence intensity of each pH-solution and AβpH-concentration in relative fluorescence units (RFU) was plotted using GraphPad Prism software.
10
Islet Viability Assessment via Fluorescence
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Prior to transplantation, encapsulated islets were stained for viability. Cells were incubated in calcein AM (4 μM, ThermoFisher) and propidium iodide (1 μg/mL, ThermoFisher) for 30 minutes. Fluorescence was captured with a Cytation 5 Imaging Multi-Mode Reader (Biotek Instruments). Using the Cytation software, the area of calcein-stained (live) cells was divided by the total cell area (obtained with brightfield images of the same fields) resulting in the percentage of live cells. Following encapsulation, microspheres were incubated in calcein AM (4 μM, ThermoFisher) and propidium iodide (1 μg/mL, ThermoFisher) for 40 minutes and the same procedures followed as described for unencapsulated cells.
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