Micrographs were captured with a Nikon Eclipse E600 darkfield microscope (Nikon Canada, Mississauga, Ontario, Canada) with a ×40 objective (×400 magnification) or a Cytation 5 Imaging Reader (BioTek) with a ×20 objective (×200 magnification). As previously described (Lithgow et al., 2020 (link)), for T. pallidum adhesion assays, samples were blinded and the numbers of endothelial nuclei (DAPI; 10–60 μm) and T. pallidum cells (FlaA; 2–8 μm) were measured from each field of view with the Cytation 5 software using size exclusion quantification settings (BioTek). Mean fluorescence intensity of VE-cadherin was quantified using the Cytation 5 Imaging Reader (BioTek) with a ×20 objective at five random fields of view per well.
Cytation 5 imaging reader
Manufactured by Agilent Technologies
Sourced in United States
The Cytation 5 is a modular, multi-mode plate reader that combines automated digital microscopy and microplate detection capabilities. It provides quantitative digital imaging and powerful multi-mode detection for live-cell analysis, endpoint assays, and high-content screening applications.
Automatically generated - may contain errors
Lab products found in correlation
Gen5 3, by Agilent Technologies (5 mentions)
96 well plate, by Corning (4 mentions)
Transit 293, by Mirus Bio (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tetrazolium dye mts cell viability assay kit, by Promega (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Biospa 8 automated incubator, by Agilent Technologies (2 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Size exclusion quantification settings, by Agilent Technologies (2 mentions)
Cytation 5, by Agilent Technologies (2 mentions)
Prestoblue cell viability assay, by Thermo Fisher Scientific (2 mentions)
96 well clear round bottom ultra low attachment microplate, by Corning (2 mentions)
Celltiter glo 2.0 reagent, by Promega (2 mentions)
Thiol fluorescent probe 4, by Merck Group (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
183 protocols using cytation 5 imaging reader
1
Quantification of T. pallidum Adhesion
2
Anti-HIV-1 Compound Potency Assay
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The anti-HIV-1 activity of compounds was examined in TZM-GFP cells. The potency of HIV-1 inhibition was determined based on the inhibition of viral LTR-activated GFP expression in the presence of compounds compared to DMSO controls. Briefly, TZM-GFP cells were plated at a density of 1 × 104 cells per well in a 96-well plate. After 24 h, media was replaced with increasing concentrations of compound. Cells were exposed to HIV-1NL4-3 (MOI = 1) 24 h post treatment. After 48 h incubation, anti-HIV-1 activity was determined by counting the amount of GFP-positive cells on a CytationTM 5 Imaging Reader (BioTek, Winooski, VT, USA), and 50% effective concentration (EC50) values were determined. All cell-based assays were conducted in duplicate and in at least two independent experiments. Final values were calculated for each independent assay, and average values for all assays were calculated. Cells were observed periodically by microscope during the antiviral assays, and cell apoptosis was observed during the course of the assays, suggesting significant cytotoxicity effects from the compounds.
3
Quantifying Tissue Oxidative Stress
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For the detection of ROS in tissue samples, OxiSelectTM ROS/RNS In Vitro Assay Kit (#STA-347, Cell Biolabs, San Diego, CA, USA) was used. In brief, 10 mg/mL of tissue was homogenized in ice-cold 1xPBS and centrifuged at 10,000× g for 5 min at 4 °C right after extraction. Homogenates were stored at −80 °C until use. The assay was performed according to the manufacturer’s instructions. In brief, 50 µL of each sample was plated in triplicates in a 96-well plate suitable for fluorescence measurement. Then, 50 µL of catalyst was added to each well and incubated for 5 min at room temperature. Afterward, 100 µL of 2′-7′dichlorofluorescin diacetate (DCFH) solution was added to each well. Fluorescence was measured after an incubation of 20 min at room temperature, protected from light, using a CytationTM 5 imaging reader (BioTek Instruments, Winooski, VT, USA) at 480 nm excitation and 530 nm emission with a reading time of 100 ms. ROS assay was conducted with tissue samples collected from four wild-type and four wobbler mice.
4
Nanoemulsion Cytotoxicity Assay in NHDF and 3T3 Cells
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Approximately 20,000 NHDF cells and 10,000 3T3 cells were seeded in 96 well plates and grown for 24 h at 37 °C and 5% CO2 in 100 µL of the corresponding cell culture media shown in Table 3 . After adding 50 µL aseptic and 0.2 µm of the sterile, filtered and differently diluted nanoemulsions, the cells were incubated for 4 or 24 h. The cell viability was determined by a resazurin reduction assay. Therefore, 30 µL of a 0.15 mg/mL resazurin solution was added and the mixture was incubated for 2 h. Then, the fluorescence intensity was determines with the CytationTM 5 imaging reader (BioTek Instruments) using the RFP 531(excitation)/593(emission) filter set. The cell viability was expressed as a percentage of the negative controls (untreated cells) after subtraction of the blank. All experiments were conducted in triplicate. The mean inhibitory concentration IC50 was determined by linear interpolation.
5
Quantifying Osteoclast Formation
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Adherent cells from the precommitment and continuous RANKL assays were fixed in a solution containing 4% formaldehyde and 0.05% Triton X-100 solution in 1× PBS for 10 min and then in a 1:1 acetone:ethanol solution for 1 min. The fixed cells were then TRAP-stained with reagents from the Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma, number 378A). The TRAP-stained wells were also stained with DAPI to visualize the nuclei. Entire wells were imaged at 4× magnification using a Biotek Cytation 5 Imaging Reader (Agilent, Winooski, VT), and then the images were stitched together. The stitched color brightfield and DAPI images were merged and blinded prior to osteoclast quantification. Osteoclasts, defined as TRAP+ cells with 3 or more nuclei, were enumerated from images using Fiji ImageJ (70 (link)).
6
Antibiotic Synergy Evaluation for P. aeruginosa
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Growth curves for P. aeruginosa PAO1 (K767) and PAO1∆mexXY (K1525) were measured in the absence or presence of amikacin or tobramycin at concentrations ranging from ¼ to 2× the MIC and with select berberine ligands (64 µg/mL). Berberine ligands were prepared and added to 96-well microtiter plate as described above for the checkerboard assays. Microtiter plates containing technical replicates for each antibiotic-berberine pair were incubated for 18 hours with vigorous shaking using in Cytation5 Imaging Reader (Agilent BioTek). OD600 values were recorded every 15 minutes and select time points (0, 2, 4, 6, 8, 10, 12, 18, and 24 hours) were plotted. Growth curves were performed in duplicate at least twice from freshly streaked plates on separate days.
7
Rapid CRISPR-Cas12a Detection Assay
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TwistAmp@ Basic kit was purchased from TwistDx™. The RPA primers, crRNA, AsCas12a, and fluorophore-quencher probes were all obtained from Integrated DNA Technologies, and detailed information about the synthetic oligonucleotides are listed in Table 2 . The RPA primer sets were designed using PrimerQuest™ Tool. Additionally, NEBuffer™ r2.1 was purchased from New England Biolabs. The RPA reaction was conducted based on the instructions: A mixture of 29.5 μL of rehydration buffer, 11.2 μL of nuclease-free water, and 2.4 μL each of forward and reverse primers (10 μM) was added to the enzyme pellet. Then, 2 μL of purified DNA and 2.5 μL of MgOAc (280 mM) were added and mixed to achieve a total volume of 50 μL. The mixture was incubated at 37 °C for 20 min. Following the incubation, 2 μL of RPA amplicons were added to a pre-assembled CRISPR-Cas12a mixture comprising 50 nM of AsCas12a, 62.5 nM of crRNA , 10× buffer, and 2.5 μM of ssDNA-FQ probe, resulting in a final reaction volume of 20 μL. The reaction solution was incubated at 37 °C for 30 min. After the incubation, the mixture was excited by a blue light transilluminator (brand: SmartBlue, Part number: NEB-E4100, excitation wavelength of 465 nm) for naked-eye observation. Finally, 20 μL of nuclease-free water was added to 5 μL of the mixture, which was then characterized by an Agilent BioTek Cytation 5 imaging reader.
8
HFRS Serum Neutralization Assay
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The indicated dilutions of HFRS convalescent serum samples were incubated with 100 plaque forming units (PFUs) of rVSV-HTNV-GP for 1 h at 37 °C. Serum-virus complexes were added to Vero E6 cells in 96-well plates and incubated at 37 °C for 7 h. Subsequently, cells were fixed in 4% PFA and stained with Hoechst 33258. Images were acquired with a BioTek Cytation 5 imaging reader (Agilent, California, USA) in FITC channel to visualize GFP-positive cells. Data were processed using Prism software (GraphPad 8.0, Inc., La Jolla, CA, USA).
9
Growth Curves of P. aeruginosa with Antibiotics and Berberine
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Growth curves for P. aeruginosa PAO1 (K767) and PAO1ΔmexXY (K1525) were measured in the absence or presence of amikacin or tobramycin at concentrations ranging from ¼ to 2× the MIC and with select berberine ligands (64 μg/mL). Berberine ligands were prepared and added to 96-well microtiter plate as described above for the checkerboard assays. Microtiter plates containing technical replicates for each antibiotic-berberine pair were incubated for 18 hours with vigorous shaking using in Cytation5 Imaging Reader (Agilent BioTek). OD600 values were recorded every 15 minutes and select time points (0, 2, 4, 6, 8, 10, 12, 18, and 24 hours) were plotted. Growth curves were performed in duplicate at least twice from freshly streaked plates on separate days.
10
Quantification and Western Blot Analysis of IGF2BP3
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LPS2 cells were lysed with SDS buffer, and protein lysates were quantified using a BCA Protein Assay Kit (ThermoFisher, West Hills, CA, USA; Cat#23225) following the manufacturer’s instructions. Sample analysis was completed using the spectrophotometer settings at 562 nm on a Cytation 5 Imaging Reader (Agilent Technologies, Santa Clara, CA, USA). Protein lysates were electrophoresed using 4–12% Bis-Tris gels. Resolved proteins were transferred to a PVDF membrane. The following primary antibodies were used: IGF2BP3 (Cell Marque, Cat# 433R, EP286, 1:1000), IGF2BP3 (MBL International Corp, Woburn, MA, USA; Cat# N009P, 1:1000) and Beta-actin (Cell Signaling Technology, Danvers, MA, USA; Cat# 3700S, 1:2000), which served as a loading control. Bound antibodies were visualized using ImobilonTM Western (Millipore Corporation, Billerica, MA, USA).
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