NCTC clone 1469 and BRL-3A cell lines were inoculated into 96-well plates with a concentration of 1 × 104 cells per well and incubated with a variety of concentrations (12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 400 μg/mL, 800 μg/mL, 1600 μg/mL, and 3200 μg/mL) of ASP and AMP for 7 days. Cell viability was determined by a AlamarBlue® Cell Viability Assay Kit (AB, DAL1100, Invitrogen Corporation, Waltham, MA, USA) on day 1, day 3, day 5, and day 7. The cells were supplemented with AB solution (medium199: fetal bovine serum: AB = 8:1:1 v/v) after the removal of the medium and PBS washing. For 4 h of incubation, the absorbance (optical density, OD) at 570 nm was measured and calculated according to the formula: Cell viability (%) = (OD experimental − OD blank)/(OD control − OD blank) × 100%. Experiments were performed in triplicate.
Alamarblue cell viability assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AlamarBlue Cell Viability Assay Kit is a fluorometric and colorimetric assay used to quantitatively measure cell proliferation and cytotoxicity. It utilizes the active ingredient resazurin, which is a non-toxic, cell-permeable compound that is blue in color and non-fluorescent. When introduced into the cell culture, resazurin is reduced by metabolically active cells, changing the color to red and becoming highly fluorescent. This color change and fluorescence can be measured to determine the number of viable cells present.
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Lab products found in correlation
Safire2 multi detection microplate reader, by Tecan (3 mentions)
Polarstar omega microplate reader, by BMG Labtech (2 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Glomax, by Promega (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Mtt stock solution, by Merck Group (1 mentions)
El800 microplate reader, by Agilent Technologies (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tetrahydrofuran, by Merck Group (1 mentions)
2 hydroxyethyl methacrylate, by Merck Group (1 mentions)
Dibutyltin dilaurate, by Merck Group (1 mentions)
Isophorone diisocyanate, by Merck Group (1 mentions)
Alizarin red staining kit, by Cyagen (1 mentions)
Live dead assay kit, by Thermo Fisher Scientific (1 mentions)
Alkaline phosphatase alp kit, by Beyotime (1 mentions)
12 protocols using alamarblue cell viability assay kit
1
Cytotoxicity Evaluation of ASP and AMP
2
Quantification of Cell Death Assays
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Cell death was typically measured using trypan blue exclusion assay to detect plasma membrane integrity as previously described [54 ]. For quantification of cell death rate, cells were trypsinized and stained with trypan blue followed by counting with a hemocytometer under microscope. In some experiments, propidium iodide staining was performed. Red-stained cells were considered dead. Quantification of cell death was further confirmed using flow cytometry. Cell viability was typically assessed in 96-well format with Alamar Blue Cell Viability Assay Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
3
Cell Viability Assay with AlamarBlue
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Cell viability was determined using the alamarBlue Cell Viability assay kit (Invitrogen™, Thermo Fisher Scientific Inc., Waltham, MA, USA) as previously described [33 (link)]. Briefly, the cells (1 × 104 cells/well) were seeded into 96-well plates for 24 h. After treatment with 4-AAQB or alendronate (ALN) for 72 h, an alamarBlue solution was added for 2 h at 37 °C. The absorbance was measured at 570/630 nm using a spectrophotometer (Spectra Max 190; Molecular Devices, Sunnyvale, CA, USA).
4
Evaluating Cell Viability with AlamarBlue
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Cell viability was determined with the alamarBlue cell viability assay kit (Molecular Probes, Eugene, Oregon, USA) following the manufacturer’s instructions. Briefly, SH-SY5Y cells were seeded onto 96-well culture plates at a 4 × 104 cell per well density and allowed to adhere overnight. Various concentrations (0, 5, 10 and 20 μg/mL) of PEG-SWNT were added into cells with or without 50 μM of 6-OHDA, and cells were allowed to incubate for 24 hours. PEG-SWNTs were pretreated 1 hour prior to 6-OHDA treatment or treated with 6-OHDA simultaneously. At the end of the experiment, 10% v/v alamarBlue (10 μL) was added into each well and fluorescent intensity was measured (excitation 530 nm, emission 590 nm) using a fluorescence plate reader (Glomax, Promega; Madison, Wisconsin, USA).
5
Proliferative Potential of hUCB-MSCs Evaluated
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The proliferative potential of the hUCB-MSCs was evaluated based on the ability of live cells to convert a tetrazolium salt into purple formazan using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assay. hUCB-MSCs, HUVECs, iNSCs (20,000 per well) were seeded on 24-well plates. After 24 h of incubation with or without GQDs, 50 mL MTT stock solution (5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was added to each well, and the plates were further incubated for 4 h at 37 °C. The supernatant was removed, and 500 μL of DMSO (dimethyl sulfoxide) was added to each well to solubilize the purple formazan crystals. The solution was then transferred to a 96-well microplate for measurement. The absorbance at a wavelength of 540 nm was measured using an EL800 microplate reader (BIO-TEK Instruments, Winooski, VT, USA). All of the measurements were performed in triplicate. AlamarBlue cell viability assay kit was also utilized to assess additional neuron viability (Cat#: DAL1025; Molecular Probes™), following the manufacturer’s instructions.
6
Cytotoxicity Evaluation via AlamarBlue Assay
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Cell viability was evaluated with an alamarBlue Cell Viability Assay Kit (#88952, Thermo Fisher Scientific Inc. Rockford, IL, USA) according the manufacturer’s instructions. Described briefly, cells were plated in a 96-well plate and exposed to various concentrations of the cytotoxic compounds for an indicated time. The alamarBlue Reagent (10 µl) was added to each well, incubated at 37°C in 5% CO2 for four hours, and then the plates were measured at 545nm/590nm (Ex/Em) using the Tecan Safire2 Multi-detection Microplate Reader (Morrisville NC, USA). Average percentage of inhibition at each concentration was calculated as previously described.
7
Cell Viability Assay Protocol
8
Cytotoxicity Evaluation via AlamarBlue Assay
9
AlamarBlue Cell Viability Assay
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Cell viability assay was conducted using AlamarBlue cell viability assay kit (ThermoFisher) following manufacturer’s protocol. Cells were cultured in Corning 96-well black plates with clear bottom (5000 cells/well). Drugs were added to cells for 72 hour following detection using POLARstar Omega Microplate Reader (excitation at 544nm and emission at 590nm) (BMG LABTECH, Germany).
10
Evaluating Erastin's Cell Viability Impact
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Cell viability was evaluated using the AlamarBlue Cell Viability Assay Kit (Thermo Fisher, IL, USA) according to the manufacturer’s instructions. The growth inhibition rate presented reflects the growth inhibitory effect of erastin treatment on cells compared to the cells without erastin treatment. The growth inhibitory rate is obtained as follows: growth inhibition rate (%)=100%-(viability of the indicated group with erastin treatment)/(viability of the control group without erastin treatment) × 100%. For the control group without erastin treatment, the inhibition rate is 0.
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