H460 cells were seeded in 96 well plates (5000 cells/well) in 10% serum RPMI. The next day, the cells were transfected with siRNA directed against RSK1/2/3 (siGenome, Dharmacon) using DharmaFECT 1 transfection reagent (Dharmacon). After 48 hours, the cells were starved in serum free RPMI for 6 hours prior to treatment with TNFα (0–1000 ng/ml). Cell Viability was measured after 24 hours of TNFα treatment using the CellTitre Blue assay (Promega). To verify siRNA knockdown of RSK1, 2 and 3 protein, whole cell lysates were harvested at 48 hours post transfection, run on SDS PAGE gels, transferred to nitrocellulose membrane and probed using isoform specific antibodies: RSK1 (#9333), RSK2 (#9340) and RSK3 (#9343, all from Cell Signaling). Secondary antibodies used were anti-rabbit-HRP and anti-mouse-HRP (GE healthcare). Blots were stripped and re-probed using a β-actin mouse polyclonal antibody (Santa Cruz) as a loading control.
Celltitre blue assay
Manufactured by Promega
Sourced in United Kingdom
The CellTiter-Blue Cell Viability Assay is a fluorometric method for estimating the number of viable cells present in a multi-well format. The assay uses a redox indicator dye that changes color in response to cellular metabolism, allowing for the quantification of viable cells in a simple, one-step protocol.
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Lab products found in correlation
Modulus 2 9300 062, by Promega (2 mentions)
β actin mouse polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (1 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (1 mentions)
Prism software, by GraphPad (1 mentions)
Prism 5, by GraphPad (1 mentions)
Phenol red free dmem media, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Click it edu assay, by Thermo Fisher Scientific (1 mentions)
Hcs nuclear mask blue, by Thermo Fisher Scientific (1 mentions)
96 well culture plate, by Thermo Fisher Scientific (1 mentions)
Apo one caspase 3 7 assay, by Promega (1 mentions)
Allstars scramble, by Qiagen (1 mentions)
10 protocols using celltitre blue assay
1
RSK Knockdown Effects on TNFα Response
2
Cell Viability and Proliferation Assays
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Cells were seeded into 96-well tissue culture-treated plates at 2.0 × 104 cells/mL for up to 24 h. DMSO stock solutions of compounds were serially diluted first in DMSO and subsequently into RPMI before being added to the cell cultures in triplicates, with 10 μM being the highest tested concentration. The plates were incubated for 72 h as above. The CellTitre-Blue assay (Promega, Madison WI) was then added to the cultures and incubated a further 4 h before the fluorescence measured following the manufacturers recommendations. The fluorescence data from compounds were normalized to that DMSO and the resulting data fitted to a non-linear regression equation to obtain IC50 using GraphPad Prism 5.0 Software (La Jolla, CA, USA).
To determine the effect of HSN431 on actual leukemia cell counts, MV4–11 cells were seeded into 96 well plates as above and treated with either DMSO vehicle, or HSN431 at the following concentrations: 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, and 10 nM. Cells were incubated for 72 h, and then counted using trypan blue exclusion on the Countess automated cell counter (Life Technologies, Carlsbad, CA). Cell counts were performed in triplicates, and the averages, standard deviations and t-test statistical analyses were graphed using GraphPad Prism software (GraphPad, La Jolla, CA).
To determine the effect of HSN431 on actual leukemia cell counts, MV4–11 cells were seeded into 96 well plates as above and treated with either DMSO vehicle, or HSN431 at the following concentrations: 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, and 10 nM. Cells were incubated for 72 h, and then counted using trypan blue exclusion on the Countess automated cell counter (Life Technologies, Carlsbad, CA). Cell counts were performed in triplicates, and the averages, standard deviations and t-test statistical analyses were graphed using GraphPad Prism software (GraphPad, La Jolla, CA).
3
Compound Screening in Cell Viability Assay
4
Measuring Cell Viability with AB1-42
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The viability of the cultures after treatment with Aβ1-42 was determined using the Cell-titre Blue assay (Promega, Southampton, UK). After experimental treatment, medium was removed from the wells of the 12-well cell-culture plate. Subsequently, the plate was washed with 500 μL phenol red-free DMEM media (Life Technologies), supplemented with 10% heat inactivated fetal bovine serum (NT2.N/A, NT2.A) or 10% horse serum (primary cortical cultures), 100 units/mL penicillin and 100 μg/mL streptomycin and 2 mmol/L L-glutamine. In all, 1 mL of Cell-titre blue reagent was mixed with 10 mL of the DMEM. In all, 500 μL of this solution was added to each well of the plate. The plate was incubated for 3 hours at 37°C. After incubation, medium was transferred to a 96-well plate and the absorbance was measured at 590 nm using a Thermo multiscan EX 96-well plate reader (Thermofisher, Loughborough, UK).
5
Cell Viability Assay on Scaffolds
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CellTitre-Blue® assay (Promega, Southampton, UK) was used to determine cell viability according to manufacturer’s instruction on the day of each time point. Cell seeded scaffolds were washed 3 times in PBS and then a mixture of CellTitre-Blue® assay with cell culture media (1:5) was added on scaffolds to incubate for 3.5 h. Fluorescence was read using a microplate reader (Modulus II 9300–062, Turner Biosystems) at Ex 520 nm Em 580–640 nm.
6
Cell Viability Assessment using CellTiter-Blue
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Cell viability was assessed using the Cell Titre Blue assay (Promega). The assay was conducted according to the manufacturer’s instructions. Briefly, cell laden scaffolds were incubated at 37 °C and 5% CO2 in a 5:1 ratio of complete media and Cell Titre Blue reagent for 4 h. 100 µL of the resulting media solution was then measured for fluorescence at ex. 525 nm em. 580–640 nm on a Modulus II Microplate reader.
7
Cytotoxicity Assay with Apoptosis Detection
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The same immunofluorescence assay protocol as described above was used for the cytotoxicity assays, except for the following differences. One hour before fixation, 5 μM of 5-ethynyl-2′-deoxyuridine (EdU) was added to the wells and then detected using the Click-iT Edu Assay (#C-10350, Invitrogen). Cells were stained with anti-cleaved-Caspase3 (#9664, Cell Signaling) at 4 °C overnight, anti-rabit-Alexa546 secondary antibody (#A-11035, Invitrogen) at room temperature for an hour, and HCS Nuclear Mask blue (#H32720, Invitrogen) at room temperature for 15 min. For the siRNA and small-molecule kinase inhibitor experiments, we measured cell viability after 24 h TNFα treatment using the CellTitre Blue assay (Promega).
8
Cytotoxicity Assessment of Insulin-Loaded Nanoparticles
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The cytotoxicity of insulin-loaded nanoparticles was assessed by HT29 cells and C2C12 cells by using Cell-Titre Blue assay (Promega, NSW, Australia). The HT29 cells were seeded in 96-well culture plates (Nunc, Denmark) at 1 x 10 5 cells/well until the formation of cell monolayer [83] . In Dulbecco's Modified Eagle Medium (without FBS), the HT29 cells were treated with blank medium A c c e p t e d M a n u s c r i p t (negative control), free insulin (100 µg/mL), Dz13Scr (10 µg), chitosan (0.004%) and insulinloaded nanoparticles (containing 100 µg/mL insulin) for 24 hours. Also, the C2C12 cells were seeded in 96-well cell culture plate at 1 x 10 5 cells/well for 24 h to ascertain cell attachment [84] .
The C2C12 cells were exposed to the blank medium (negative control), free insulin (100 nM), Dz13Scr (10 µg), chitosan (0.004%) and insulin-loaded nanoparticles (containing 100 nM insulin) for 24 hours. After sample treatment, the cells were washed with phosphate buffer saline. The cytotoxicity assay was conducted by the established protocol from manufacturer. The cell viability was presented in percentage in relative to non-treated cells.
The C2C12 cells were exposed to the blank medium (negative control), free insulin (100 nM), Dz13Scr (10 µg), chitosan (0.004%) and insulin-loaded nanoparticles (containing 100 nM insulin) for 24 hours. After sample treatment, the cells were washed with phosphate buffer saline. The cytotoxicity assay was conducted by the established protocol from manufacturer. The cell viability was presented in percentage in relative to non-treated cells.
9
P2RX7 Knockdown Impacts Cell Survival
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A total of 200,000 cells were seeded in 6-well plates 16 h before transfection with 70 nM of relevant P2RX7-targeted siRNA from Origene using Lipofectamine RNAiMax. The siRNA target sequences were: siRNA A: 5′-CCCGCAGAGCAAAGGAATTCAGACC-3′, B: 5′-GAGATATTGTGAGGACAAATTGAGA-3′, C: 5′-ACAATGTTGAGAAACGGACTCTGAT-3′. AllStars scramble from Qiagen was used as a control. siRNA effect on cell survival was analysed using 2000 cells seeded in 96-well plates and transfected with 70 nM siRNA. 72 h after transfection, cell numbers were assessed using the CellTitre-Blue assay (Promega) or Apo One Caspase 3/7 assay (Promega).
10
Cell Viability Assay using CellTiter-Blue
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Cell metabolism was assessed using a CellTitre-Blue ® assay (Promega, UK). Scaffold were washed 3 times in PBS and 80 µl of CellTitre-Blue ® assay was added to 400 µl cell culture media (1:5) and incubated for 2 hours. Fluorescence was read using a microplate reader (Modulus II 9300-062, Turner Biosystems) at Ex 520 nm Em 580-640 nm, N ≥ 4 independent replicates.
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