The cytotoxic activity of ESI was measured using the WST-1 assay (Beyotime, Jiangsu, China). A549 and H1299 cells were treated with ESI at various concentrations for different time points, and then washed once and incubated with WST-1 at 37 °C for 2 h. The plates were read on an automated microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) at 450 nm.
Wst 1 assay
Manufactured by Beyotime
Sourced in China
The WST-1 assay is a colorimetric method for the determination of cell viability and proliferation. The assay is based on the cleavage of the tetrazolium salt WST-1 to a colored formazan dye by mitochondrial dehydrogenases in viable cells. The intensity of the color is directly proportional to the number of metabolically active cells in the sample.
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Lab products found in correlation
Automated microplate spectrophotometer, by Agilent Technologies (4 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Transwell chamber, by BD (1 mentions)
Fitc dextran, by Targetmol (1 mentions)
Synergy ht, by Tecan (1 mentions)
Infinite 200 pro microplate reader, by Tecan (1 mentions)
Inverted fluorescence microscope, by Zeiss (1 mentions)
Apoptosis detection kit, by Keygen Biotech (1 mentions)
13 protocols using wst 1 assay
1
Cytotoxicity Evaluation of ESI
2
In vitro cell migration and invasion assay
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In vitro cell migration assays were performed by using uncoated, 8 μm pore-size Transwell chambers as described previously (BD Biosciences) [24 (link)]. Cells in serum-free medium were seeded into the upper chamber. Complete medium was added to the bottom wells of the chamber. After 24 h, the cells that had not migrated were removed from the upper surface of the filters by using cotton swabs. Cells that had migrated into the lower surface were fixed with methanol and stained with crystal violet (Sigma-Aldrich). Images of five different fields were captured using microscope (Olympus, Tokyo, Japan) from each membrane, and the numbers of migrated cells were counted. The mean of values from triplicate assays for each experimental condition was calculated. A similar protocol was used to measure invasive potential with the use of BD Bio Coat Matrigel Invasion Chambers (BD Biosciences). WST-1 assay (Beyotime) was used to measure cell viability, as previously described [25 (link)].
3
NDV Infection and Inhibitor Assay
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HeLa cells were infected with NDV Herts/33 at a multiplicity of infection (MOI) of 5, or mock-treated with the same medium without virus at 37°C. Following a 1-h adsorption period, unattached viruses were removed and cells were washed with PBS and then cultured in fresh medium. Viral titers were determined on DF-1 cells as median tissue culture infective dose (TCID50 ) calculated using the Reed-Muench formula [28 (link)]. Viral titers were expressed as percentage of control. Triplicate wells were tested for each experiment, and the full experiment was repeated twice. Virus replication was determined by quantifying NDV P and NP protein expression, using Western blot as described previously [29 (link)].
The optimal concentrations of drugs used in this study were determined by WST-1 assay according to the manufacturer’s guidelines (Beyotime Biotechnology, Shanghai, China) (S1 Fig ). For inhibitor experiments, the 1,000× stock of inhibitors were prepared in DMSO. DMEM containing DMSO was used for the mock-treatment. Cells were pretreated with the inhibitor for 1 h, then infected with the virus for 1 h, washed with PBS for three times, and placed in serum-free media containing fresh inhibitor.
The optimal concentrations of drugs used in this study were determined by WST-1 assay according to the manufacturer’s guidelines (Beyotime Biotechnology, Shanghai, China) (
4
Cell Viability Assessment via WST-1 Assay
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The cells were seeded in 96-well plates at 5,000 cells/well and received different treatments for 72 h. Thereafter, a Water Soluble Tetrazolium (WST-1) assay (Beyotime Biotechnology, Shanghai, China) was employed to evaluate cell viability in accordance with the manufacturer's instructions. Absorbance was measured at 450 nm using a multimode plate reader. The data were used to determine cell viability rates.
5
Cell Proliferation Assay using WST-1
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We used the WST-1 assay (Beyotime) to examine the cell proliferation ability as we previously described (32 (link)). In brief, 2000 cells were seeded in 96-well plates with final volumes of 100 μl per well. Cells were treated with 10 μl WST-1 solution and continuously incubated for 2 h at 37°C. The absorbance was detected by a microplate reader (BioTek, Vermont, Winooski, VT, USA) using the dual wavelength method by OD450–OD630 (nm).
6
Cell Proliferation Assay for miRNA
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Proliferation assays were conducted using WST-1 assay (Beyotime, Shanghai, People’s Republic of China). After CCLP1 and SG-231 cells were transfected with miR-10-a-5p mimic or miR-10a-5p inhibitor or scramble control for 6 hours, cells were seeded in 96-well plates (2,000 cells/well). At 0, 24, 48, and 72 hours, culture medium was removed and 100 µL fresh medium containing 10 µL of WST-1 reagents was added into the wells. After 2–3 hours, the absorbance was measured at 450 nm by using ELISA Microplate Reader (Biocompare, San Francisco, CA, USA).
7
Cytotoxicity and Cellular Uptake of NAP
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WST-1 assay (Beyotime) was used to determine the cytotoxicity of NAP. In brief, A549 cells (2 × 103) were seeded in 96-well plate and treated with increasing concentration of NAP (up to 400 μM) for 48 h. WST-1 was added and the plate was read after 1.5 h on an automated microplate spectrophotometer (BioTek Instruments, Vermont, USA) at 450 nm according to the manufacturer’s instructions.
Colony-formation assay was performed to detect long-term survival of A549 cells in NAP treatment. 400 cells seeded in 12-well plates were treated with indicated concentration of NAP for 15 days. The plates were washed twice with PBS and fixed with methanol for 10 min at room temperature and then stained with 1% crystal violet for 5 min. The number of colonies was counted using ImageJ software (version 1.44I).
Cellular uptake assay was performed by using FITC-Dextran (TargetMol). A549 cells were seeded onto 12-well plate and incubated with FITC-Dextran (0.1 mg/mL) for 24 h, then the cells were treated with 50, 100, 150 and 200 μM of NAP for 24 h and 48 h, respectively. The fluorescence (excited at 488 nm and emitted at 530 nm) was measured with a multifunctional microplate reader Spark 10 M (Tecan, Synergy-HT).
Colony-formation assay was performed to detect long-term survival of A549 cells in NAP treatment. 400 cells seeded in 12-well plates were treated with indicated concentration of NAP for 15 days. The plates were washed twice with PBS and fixed with methanol for 10 min at room temperature and then stained with 1% crystal violet for 5 min. The number of colonies was counted using ImageJ software (version 1.44I).
Cellular uptake assay was performed by using FITC-Dextran (TargetMol). A549 cells were seeded onto 12-well plate and incubated with FITC-Dextran (0.1 mg/mL) for 24 h, then the cells were treated with 50, 100, 150 and 200 μM of NAP for 24 h and 48 h, respectively. The fluorescence (excited at 488 nm and emitted at 530 nm) was measured with a multifunctional microplate reader Spark 10 M (Tecan, Synergy-HT).
8
Cell Proliferation Assay using WST-1
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Cell proliferation was determined by WST-1 assay (Beyotime Institute of Biotechnology, Inc.). Briefly, 5×103 HMSCs were planted into a well of 96-well plate and cultured at 37°C for 24 h. According to the manufacturer's instructions, 20 µl WST-1 were added to 200 µl cell culture medium and incubated at 37°C in the dark for 2.5 h. OD450 and OD630 were measured using a microplate reader (BioTek Instruments, Inc.).
9
Proliferation Assay of Prostate Cancer Stem Cells
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Proliferation assays were conducted using WST-1 assay (Beyotime, Shanghai, China). ALDH+CD44+CXCR4+CD24+ cells were isolated and purified from LNCaP xenograft tumor and seeded in 96-well plates and treated with peptide TAP1 or control peptide at 1, 2, 5, 10, 20, and 40 μM for 72 h. ALDH+CD44+CXCR4+CD24+ cells were incubated with peptide TAP1 (20 μM) or control peptide (20 μM) for 24, 48, and 72 h. ALDH+CD44+CXCR4+CD24+ cells and ALDH−CD44−CXCR4−CD24− cells were incubated with anti-androgens or chemotherapeutic agents at an indicated concentration and with peptide TAP1 (5 μM) or control peptide (5 μM) for 72 h. Culture medium was removed, and 100 μl fresh medium containing 10 μl of WST-1 reagents was added into the wells. After 1–2 h, the absorbance was measured at 450 nm using an ELISA Microplate Reader (Biocompare, San Francisco, CA).
10
Evaluating GRO-γ Effects on HTR-8/SVneo Cells
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HTR-8/SVneo cells, which were seeded into 96-well microtiter plates at 1×103 cells/well concentration, were treated with Recombinant Human GRO-γ (rhCXCL3) at different concentrations and time gradients after 12 h of starvation. The proliferating activities of these cells were measured via water-soluble tetrazolium salt (WST-1) assay (Beyotime, China). About 10 µL of WST-1 was added per well. Moreover, the cells were incubated for 1 h before their optical density was analyzed using an Infinite 200 PRO microplate reader (Tecan Group Ltd., Switzerland) at 450 nm to determine cell viability. Each test was repeated in triplicate.
Apoptosis was assessed via staining by using annexin-V-conjugated fluorescein isothiocynate (FITC) and propidium iodide (PI) in an apoptosis detection kit (KeyGEN BioTECH, China). The cells were treated with different concentrations of recombinant proteins after being seeded on a slide for 24 h. Afterwards, the cells were washed with PBS twice and dyed in 500 µL of the binding buffer with 5 µL of Annexin V-FITC and 5 µL of PI for 5 min at room temperature. Finally, cell apoptosis quantification was performed using an inverted fluorescence microscope (Zeiss, Germany).
Apoptosis was assessed via staining by using annexin-V-conjugated fluorescein isothiocynate (FITC) and propidium iodide (PI) in an apoptosis detection kit (KeyGEN BioTECH, China). The cells were treated with different concentrations of recombinant proteins after being seeded on a slide for 24 h. Afterwards, the cells were washed with PBS twice and dyed in 500 µL of the binding buffer with 5 µL of Annexin V-FITC and 5 µL of PI for 5 min at room temperature. Finally, cell apoptosis quantification was performed using an inverted fluorescence microscope (Zeiss, Germany).
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