Cell viability was determined using the WST--1 reagent (Cell Count Reagent; Nacalai-Tesque) according to the manufacturer’s protocol. Target cells (ARPE-19 or hCMEC/D3) were plated at 1.5 × 104 cells/well in 96-well microtiter plates. ARPE-19 cells were treated for 24 h with NF-κB inhibitor or cytokines, or hCMEMC/D3 cells were treated with diluted conditioned media for 24 h. Cell viability reagent (10 μL) was added to each well for 4 h, and then, the resulting color change was measured using a Tecan-2000 plate reader at 450 nm wavelength. Relative changes in absorbance were determined after subtracting the absorbance measured in cell-free wells. Cell viability was then calculated as percent changes relative to DMSO-treated or control media-treated control cells.
2000 plate reader
Manufactured by Tecan
Sourced in Switzerland
The Tecan -2000 plate reader is a versatile laboratory instrument designed for various absorbance-based assays. It can measure absorbance across a wide range of wavelengths, enabling researchers to perform multiple types of assays within a single platform.
Automatically generated - may contain errors
Lab products found in correlation
Cell count reagent, by Nacalai Tesque (1 mentions)
C57bl 6n, by Jackson ImmunoResearch (1 mentions)
Fvb nj, by Jackson ImmunoResearch (1 mentions)
Wst 1, by Nacalai Tesque (1 mentions)
X tremegene hp dna transfection reagent, by Merck Group (1 mentions)
Pcdnatm3.1, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Hiperfect, by Qiagen (1 mentions)
Celltiter glo one solution assay, by Promega (1 mentions)
Prism 8, by GraphPad (1 mentions)
5 protocols using 2000 plate reader
1
Cell Viability Assay with WST-1 Reagent
2
Biodistribution of Rho-acNPs in Mice
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All procedures were performed using pre-chilled equipment and solutions. Biodistribution studies were done in FVB/NJ (Jackson Laboratory, Strain # 001800) and C57BL/6N mice (Jackson Laboratory, Strain # 005304). In FVB/NJ mice, saline (vehicle) or Rho-acNPs were injected three times (one injection every other day) into the mice. The organs (e.g., liver, kidney, brain, white adipose tissue, heart, lungs) were harvested following 48 h of the last injection and rinsed in PBS. The tissues were weighed, minced, and suspended in 6 mL of PBS. The preparation was then mechanically homogenized with 9 strokes in glass-Teflon dounce homogenizer. The total protein content from each tissue was measured, and 2 µg, 10 µg, 20 µg, and 100 µg of the protein from different tissues was used to assess for linear range of rhodamine fluorescence using Tecan 2000 Plate-reader at (Ex/Em: 553/627 nm). For the comparison between different organ tissues, 10 µg of protein per tissue were used, and fluorescence signal is expressed as fold change to saline control.
3
Microtiter Assay for Oxidative Stress
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For assessing responses to oxidative stress caused by hydrogen peroxide or cobalt chloride, a microtiter assay was used. Using 96-well-tissue culture microtiter plates, 1.5 × 104 cells/well were plated in 100 μl of media. After attachment, media was exchanged for differentiation media and cells were treated for 7 days to develop a neuronal phenotype. For assessing treatments, media was exchanged for DMEM + 1% FBS to which different concentrations of hydrogen peroxide (0–250 μM) or cobalt chloride (0–200 μM) were added. Cells were incubated for 24 h and then 10 μl of cell viability reagent WST-1 (Cell Count Reagent, Nacalai-Tesque, Japan) was added to each well. Absorbance at 450 nm was recorded using a Tecan 2000 plate reader after 1, 2 and 4 h of further incubation. Changes in absorbance were calculated after subtraction of absorbance from cell-free wells. Degree of protection was calculated as percentage changes relative to untreated cells for each clone.
4
Reverse Transfection of Cells with Plasmid and siRNA
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Cells were reverse transfected with either plasmid DNA or siRNA. Plasmid transfection was performed using the X-tremeGENE HP DNA transfection reagent (Merck, Germany) according to the manufacturer’s instructions. In brief, 100 µL transfection mix containing serum-free RPMI-1640 cell culture medium, 2 µg plasmid DNA, and 2 µL transfection reagent was incubated for 15 min at room temperature and directly added to 4 × 105 cells in 2 mL of medium after seeding. The TYRO3 and the control expression vectors were purchased from OriGene, USA and Thermofisher, USA (TYRO3:pCMV6-XL4, CAT#: SC108283, pcDNATM3.1(+), CAT#: V790-20). For siRNA transfection, HiPerFect (Qiagen, Germany) was used according to the manufacturer’s protocol. Briefly, a transfection mix of 100 µL serum-free RPMI-1640, 100 nM siRNA, and 12 µL transfection reagent was incubated for 5 min and added to 4 × 105 cells in 2.3 mL medium and incubated for 24 h before treatment. siRNAs were purchased from Qiagen, Germany (SI00288344, SI00288351). Cell viability was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer’s recommendations. Absorbance was measured using a Tecan Plate Reader 2000 (Tecan, Switzerland).
5
Cell Viability Determination via CellTiter Glo
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Cell viability was determined using CellTiter Glo One Solution Assay (Promega, USA) according to the manufacturer’s recommendations. Luminescence was measured using a Tecan Plate Reader 2000 (Tecan, Switzerland). Relative IC50 was generated within Prism 8 (GraphPad Software, LCC) by normalizing single measurements, log transform drug concentration, and performing a dose-response curve fitting. IC50 values were calculated from at least three biological replicates using nonlinear regression algorithms.
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