Cell-based high-content screening (HCS) multi-parameter cytotoxicity analysis [Thermo Scientific Cellomics ®ArrayScan ® VTI HCS Reader (Pittsburgh, USA)] was used to measure the cell health status of GC-2 and TM-4 cells after Silica NP exposure. 5 × 104 GC-2 or TM-4 cells were separately plated in Collagen I-coated 96-well plates (BD Biocoat® Plates, No. 354407) and incubated for 24 h. After exposure to different concentrations of Silica NP (0.1, 1, 10, 100 μg/mL), control medium and positive control (120 μM Valinomycin) for 24 h, cells were stained using Cellomics® Multiparameter Cytotoxicity 3 Kit (8408102; Cellomics). Cell images and data were obtained from HCS. Four fluorescent signals were recorded. Blue nucleus images stained with Hoechst 33342; Green cell membrane stained with green permeability dye; Yellow mouse monoclonal antibody against cytochrome c; Red images stained with mitochondrial membrane potential dye. For each concentration of Silica NP, four independent wells were measured. The 20× objective was used to collect images. Enough cells (>500) were captured for the analysis, 16 fields per well were imaged. The analysis was performed by using the Automated Image and Data Analysis Software (the OSIS Scan software).
Biocoat plates
Manufactured by BD
Biocoat plates are cell culture plates with a pre-coated surface designed to enhance cell attachment and growth. They provide a standardized and consistent substrate for various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cellomics arrayscan vti hcs reader, by Thermo Fisher Scientific (3 mentions)
Cellomics multiparameter cytotoxicity 3 kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
C6 flow cytometer, by BD (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Avantor (2 mentions)
Lipofectamine 2000, by Addgene (2 mentions)
Multiparameter cytotoxicity 3 kit, by Thermo Fisher Scientific (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Gdc 0623, by Selleck Chemicals (1 mentions)
Rna lysis buffer, by Zymo Research (1 mentions)
Recombinant rat il 1β, by Thermo Fisher Scientific (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Ciliobrevin d, by Merck Group (1 mentions)
Human bdnf, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Luclite luminescence substrate reagent, by PerkinElmer (1 mentions)
Microbeta1450 liquid scintillation counter, by PerkinElmer (1 mentions)
Origin software, by OriginLab (1 mentions)
9 protocols using biocoat plates
1
Silica Nanoparticle Cytotoxicity Screening
2
High-Content Screening of Cytotoxicity
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Multi-parameter cytotoxicity was analyzed by cell-based high-content screening (HCS) analysis (Thermo Scientific Cellomics ®ArrayScan® VTI HCS Reader, Pittsburgh, USA) as described previously10 (link). Briefly, cells were plated at a density of 5 × 104 cells/ml in Collagen I-coated 96-well plates (BD Biocoat® Plates) and incubated for 24 h with 50 μg/ml THS, control, or 120 μM valinomycin as positive control. Then, the cells were fixed and stained using the Multiparameter Cytotoxicity 3 Kit (Cellomics) which tests nuclear integrity, cell membrane integrity; cytochrome c levels and mitochondrial membrane potential. A 20x objective was used to collect images. For each treatment, four independent wells were examined, and 16 fields per well were captured for analysis.
3
Celastrol Cytotoxicity Analysis in HL-60 Cells
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Cell-based high-content screening (HCS) multi-parameter cytotoxicity analysis [Thermo Scientific Cellomics®ArrayScan®VTIHCS Reader (Pittsburgh, USA)] was used to measure the cell health status of HL-60 cells after celastrol treatment. HL-60 cells were plated in Collagen I-coated 96-well plates (BD Biocoat® Plates, No. 354407) at a density of 2×104/well and incubated for 24 h. After exposure to different concentrations of celastrol (0.125, 0.25 and 0.5 μM) and control solvent for 24 h, the cells were stained using Cellomics® Multiparameter Cytotoxicity 3 Kit (8408102; Cellomics) according to the manufacturer's instruction. More details were according to previous report [46 (link)].
4
Quantification of Hemadsorption Assay
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Hemadsorption (HAD) was performed and quantified as previously described (47 (link)). Briefly, growth medium from HN-transfected 293T cell monolayers in 48-well Biocoat plates (Becton Dickinson Labware) was aspirated and replaced with 150 µl of CO2-independent medium (pH 7.3; Gibco) containing different concentrations of compounds and 1% RBC in serum-free CO2-independent medium and placed at 4°C for 30 min. The wells were then washed three times with 150 µl cold CO2-independent medium. The bound RBCs were lysed with 200 µl RBC lysis solution (0.145 M NH4Cl, 17 mM Tris HCl), and absorbance was read at 405 nm using a Spectramax M5 (Molecular Devices) microplate reader.
5
CRISPR Gene Editing in GFP-Expressing HEK293 Cells
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HEK293-GFP stable cells (GenTarget), which constitutively express Emerald GFP, served as the reporter cell line. Cells were maintained in “full serum media”: Dulbecco’s Modified Eagle’s Media plus GlutaMax (Life Technologies) with 10% (vol/vol) FBS and penicillin/streptomycin (1×, Amresco). 5 × 104 cells were plated on 48-well collagen-coated Biocoat plates (Becton Dickinson). 16–18 h after plating, cells were transfected with Lipofectamine 2000 (Life Technologies) according to the manufacturer’s protocol. Briefly, 1.5 µL of Lipofectamine 2000 was used to transfect 650 ng of total plasmid: 500 ng Cas9 expression plasmid, 125 ng sgRNA expression plasmid, and 25 ng near-infrared iRFP670 expressing plasmid (Addgene plasmid 45457)26 (link). 12 h after transfection, the media was replaced with full serum media, with or without 4-HT (1 µM, Sigma-Aldrich T176). The media was replaced again 3–4 days after transfection. Five days after transfection, cells were trypsinized and resuspended in full serum media and analyzed on a C6 flow cytometer (Accuri) with a 488-nm laser excitation and 520-nm filter with a 20-nm band pass. Transfections and flow cytometry measurements were performed in triplicate.
6
CRISPR Gene Editing in GFP-Expressing HEK293 Cells
7
Studying Necrotic Supernatant Effects
8
Rat Primary Neuron Culture and Treatments
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The use of all animals was approved by the Institutional Animal Care and Use Committee at the University of California, Irvine. Primary neuronal cultures were obtained from embryonic day 18 (E18) rat embryos. Dissociated cells were cultured on either pre-coated poly-L-Lysine 6-well plates (Biocoat plates, BD Bioscience) or poly-L-Lysine coated glass coverslips affixed to microfluidic chambers at 1 × 106 cells/9.5 cm2 and 5 × 106 cells/ml (for microfluidic chamber), respectively. All cells were maintained in Neurobasal (Gibco) supplemented with B27, penicillin/streptomycin, and Glutamax at 37 oC, 5% CO2, 95% humidity. Rat recombinant IL-1β (PeproTech) was used at a final concentration of 10 ng/ml. Human BDNF (PeproTech) was used at a concentration of 50 ng/ml and Ciliobrevin D (Sigma) was dissolved in sterile Me2SO at 100 μM and used at 1 μM.
9
Quantifying cAMP Synthesis in Receptor Cells
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EXAMPLE 9
Functional Assay-cAMP Synthesis
The ability of glucagon analogs to induce cAMP was measured in a firefly luciferase-based reporter assay. HEK293 cells co-transfected with a receptor (glucagon receptor, GLP-1 receptor or GIP receptor) and luciferase gene linked to cAMP responsive element were serum deprived by culturing 16 h in DMEM (Invitrogen, Carlsbad, Calif.) supplemented with 0.25% Bovine Growth Serum (HyClone, Logan, Utah) and then incubated with serial dilutions of either glucagon, GLP-1, GIP or novel glucagon analogs for 5 h at 37° C., 5% CO2 in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences, San Jose, Calif.). At the end of the incubation 100 microliters of LucLite luminescence substrate reagent (Perkin-Elmer, Wellesley, Mass.) were added to each well. The plate was shaken briefly, incubated 10 min in the dark and light output was measured on MicroBeta-1450 liquid scintillation counter (Perkin-Elmer, Wellesley, Mass.). Effective 50% concentrations were calculated by using Origin software (OriginLab, Northampton, Mass.
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