Cell proliferation was analyzed with XTT Cell Proliferation Kit II (Roche, Indianapolis, IN, USA). For the assay, cells were seeded at a density of 5000 cells per well in 96-well plates in six replicates. Before adding the compounds, cells were allowed to attach to the bottom of the wells for 24 h. After that, cells were treated with a range of concentrations of PBA and/or SB for 72 h. Cell viability was evaluated by the colorimetric assay XTT Cell Proliferation Kit II (Roche) and absorbance was measured at 490 nm using a Genios pro microplate reader (Tecan, Tecan Trading AG, Männedorf, Switzerland) in a 96-well plate. DMSO (0.1%) was used as a vehicle control. Fresh working solutions were prepared for immediate use. The half maximal inhibitory concentration (IC50) values of the different compounds were determined using Prism 9 (GraphPad Software, San Diego, CA, USA). Each experiment was performed three to five times.
Cell proliferation kit 2 xtt
Manufactured by Roche
Sourced in Germany, Switzerland, United States, Italy, Denmark
The Cell Proliferation Kit II (XTT) is a colorimetric assay used to measure cell proliferation and cell viability. The kit utilizes the tetrazolium salt XTT, which is reduced by metabolically active cells to form an orange-colored formazan product. The amount of formazan produced is directly proportional to the number of viable cells, which can be quantified by measuring the absorbance of the solution.
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Lab products found in correlation
96 well plate, by Sarstedt (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Glomax multi detection system, by Promega (4 mentions)
Ethanol, by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Merck Group (3 mentions)
Mercury orange, by Merck Group (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Merck Group (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Pm 100, by Thorlabs (2 mentions)
Fluostar optima, by BMG Labtech (2 mentions)
Infinite m200 spectrophotometer, by Tecan (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Cytotoxicity detection kit ldh, by Roche (2 mentions)
L glutamine, by Roche (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
130 protocols using cell proliferation kit 2 xtt
1
XTT-based cell proliferation analysis
2
Measuring Splenocyte Viability and Apoptosis
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Red blood cells from cell suspensions were removed with Red Blood Cell lysis buffer. After washing, cell pellets were resuspended in RPMI medium supplemented with 10% fecal bovine serum (FBS), 1% penicillin/streptomycin. Splenocytes from aged and young mice (1 × 106 cells/well) were treated in triplicates with ABT‐263 (1 μM, 0.1% DMSO) for 24 h in RPMI medium, washed and then stimulated with LPS (1 μg/mL). Cell viability was measured with XTT assay by following manufactures instructions (Cell Proliferation Kit II (XTT) Roche, MERCK). The colored formazan product was photometrically measured at 450 and 570 nm in a multiwell plate reader (Multiscan FC Microplate Reader). Analysis of annexin V expression was performed 4 h post‐ABT‐263 treatment.
3
Alginate-Gelatin Hydrogel Scaffold
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Low viscosity (LV, 250 cps) sodium alginates (SA) from brown algae, gelatin type B (225 g bloom), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (N-NHS), and anhydrous calcium chloride (CaCl2) were purchased by Sigma Aldrich, Milan, Italy. Deionized water was used for the preparation of the solution. In addition, 18- and 27-gauge (G) needles for the atomization process were purchased by BD, USA. As for biological assays, human mesenchymal stem cells (hMSCs, SCC034), Eagle’s alpha minimum essential medium (α-MEM), fetal bovine serum (FBS), antibiotic solution (streptomycin 100 µg/mL and penicillin 100 U/mL), L-glutamine, and cell proliferation Kit II (XTT, Roche) were purchased by Sigma Aldrich, Milan, Italy.
4
Cytotoxicity Evaluation in Neuronal Cells
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The analysis of cytotoxicity was performed with Cell Proliferation Kit II—XTT (Roche Molecular Biochemicals, Mannheim, Germany). Cytotoxicity was evaluated in neuronal cells differentiated at different concentrations of the tested compounds. Cells were seeded in 24-well plates and treated with the compounds for 24 h. Then, cells were incubated with XTT solution according to the manufacturer’s instructions. The absorbance was measured at 492 and 690 nm (Epoch Microplate Spectrophotometer, BioTek, Winooski, VT, USA), and the final result for each sample is directly proportional to the number of viable cells. The value obtained for the negative control was considered 100%.
5
Evaluating Keratinocyte Viability Responses
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Keratinocytes were treated with various concentrations of synthetic PSM Fα3, purified PSM Fα3, purified PSM α3 and bacterial supernatants for 24 h. Cell viability was evaluated using XTT and lactate dehydrogenase (LDH) assays. For XTT assay, XTT labeling mixture was added and cell viability assay was performed using the cell proliferation kit II (XTT, Roche, Basel, Switzerland) according to the manufacturer’s protocol. For LDH assay, supernatants were collected after 24 h and mixed with 500 µL of PBS – 0.1% Triton X-100 (Sigma-Aldrich). Cells were lysed with 1 mL of PBS – 0.1% Triton X-100 and sonicated for 30 s. The LDH released was measured with the Cobas® (Roche) analyzer and viability was calculated, yielding the ratio between the LDH released in supernatants and the total LDH measured in both supernatants and cell lysates.
6
Cell Viability Assay Protocol Using XTT
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Cell viability was assessed using the Cell Proliferation kit II (XTT) (Roche Diagnostics, Merck, Darmstadt, Germany) as previously described [59 (link)]. In this assay, the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) was cleaved by viable cells in order to form an orange formazan dye that is then quantified photometrically at 450 nm. Briefly, the cells (4 × 105 cells/mL) were cultured in 96-well plates for 24 h. The culture medium was then replaced by medium containing serial dilutions of the 3CLpro inhibitors, and the cells were incubated for 24, 48, 72, and 96 h. Then, XTT was added to each well and the plates were incubated for 2 h. Optical density was measured at 450 nm (reference wavelength—650 nm) using a Multiskan GO plate reader (Thermo Scientific Instruments, Waltham, MA, USA). For the quantifications, the background levels of media without cultured cells were subtracted.
7
Placental Explant Cell Viability Assay
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Cell viability was quantified using the Cell Proliferation Kit II XTT (Roche Diagnostics, Basel, Switzerland), according to the manufacturer’s instructions. Briefly, placental explants were incubated for 96 h with culture media adjusted with glucose 10 or 50 mM. On the day of the experiment, the XTT Reagent/Electron Coupling Reagent mixture was added, and the explants were incubated for 2 h at 37 °C in a humidified incubator 5% CO2–95% air. The absorbance was read at 450 nm using the microplate spectrophotometer xMark (BIO-RAD. Hercules, CA, USA). Absorbance was corrected to wells with XTT substrate and no tissue.
8
XTT-based Cell Proliferation Assay
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Cell Proliferation Kit II (XTT) (Roche Diagnostics, Mannheim, Germany) was utilized according to the manufacturer's instructions. In brief, electron-coupling reagent was mixed with XTT labeling reagent (1:50 dilution) and 50 µL of the mixture was added to the cells. Cells were incubated for a total time interval of 4 h under cell culture conditions. The absorbances of 100 µL aliquots were determined using a scanning multi-well spectrophotometer (Biorad 680, Hercules, CA, USA) with filters for 450 nm and 650 nm (reference wavelength).
9
Evaluating T Cell-Mediated Cytolysis
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Example 8
HBV s183-TCR mRNA electroporated activated or non-activated (resting) T cells were co-cultured with either HepG2.2.15 or HBV-infected HepG2-hNTCP at 1:3 and 1:1 E:T ratios (effectors calculated based on frequency of CD8+pentamer+ cells) for 24 h. HCV NS3 TCR mRNA electroporated non-activated (resting) T cells were co-cultured with Huh7 HCV replicon cells for 18 h, followed by quantification of luminescence as described above. To assess for target cell lysis after co-culture with T cells, supernatants from co-culture experiments were collected for measurement of alanine aminotransferase (ALT) after 24 h, or viability assays were performed using the Cell Proliferation Kit II (XTT) (Roche Applied Science, Mannheim, Germany). For blocking IFN-γ, 20 μg/ml purified anti-human IFN-γ or isotype control mouse IgG1 (BioLegend) was added. To block lymphotoxin (LT)α1β2, LTα2β1 and LIGHT, 1 μg/ml recombinant human LTβ receptor-Fc chimera (R&D Systems, Minneapolis, USA) was used.
10
Sodium Alginate Hydrogel Fabrication
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Sodium alginate (SA) from brown algae (≈250 cps), anhydrous calcium chloride (CaCl2), dopamine-hydrochloride (DA), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris base, buffer), and ethanol were purchased by Sigma Aldrich (Milan, Italy). To prepare the solutions, deionized water was used, while 18 G metallic needles (18 Ga) were purchased by BD, USA for the atomization process.
For in vitro assays, phosphate buffer solution (PBS), sodium dodecyl sulfate (SDS), human mesenchymal stem cells (hMSCs, SCC034), fetal bovine serum (FBS), BCA QuantiPro assay kit, bovine serum albumin (BSA), Cell Counting Kit-8 (CCK-8), Cell Proliferation Kit II (XTT, Roche, Basel, Switzerland), streptomycin, penicillin, and L-glutamine were purchased from Sigma Aldrich (Milan, Italy).
For in vitro assays, phosphate buffer solution (PBS), sodium dodecyl sulfate (SDS), human mesenchymal stem cells (hMSCs, SCC034), fetal bovine serum (FBS), BCA QuantiPro assay kit, bovine serum albumin (BSA), Cell Counting Kit-8 (CCK-8), Cell Proliferation Kit II (XTT, Roche, Basel, Switzerland), streptomycin, penicillin, and L-glutamine were purchased from Sigma Aldrich (Milan, Italy).
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