S2 cells were seeded on 12-well plates at a density of 1 million cells per well 24 h prior to transfection. Twenty-four hours after transfection, cells were scraped and seeded on 96 well plates at a density of ten thousand cells per well. Cell proliferation was measured using XTT cell proliferation assay kit (Biological Industries) according to the manufacturer’s protocol after 24, 48 and 72 h of transfection.
Xtt cell proliferation assay kit
Manufactured by Sartorius
Sourced in Israel
The XTT cell proliferation assay kit is a colorimetric assay used to measure cell viability and proliferation. It utilizes the tetrazolium salt XTT, which is reduced by metabolically active cells to produce a colored formazan product that can be quantified spectrophotometrically.
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7 protocols using xtt cell proliferation assay kit
1
S2 Cell Proliferation Assay
2
Cell Proliferation Assay for MCF7, MCF10A Cells
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Cell proliferation was monitored using an XTT cell proliferation assay kit (Biological Industries), according to manufacturer’s protocol. MCF7, MCF10A, M12, P69, stable MCF7/IGF1R KO and control (empty vector-transfected) cells were seeded at a density of 7,000 cells/ml in 96-well plates. Cells were treated with IGF1 or dansylcadaverine or both, starting 24 hr after seeding for an additional 24 hr. Sample absorbance was measured with a spectrophotometer at a wavelength of 450–500 nanometers. Reference absorbance to measure non-specific readings was measured at a wavelength of 630–690 nanometers.
3
Evaluating Cell Viability via XTT Assay
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Cells were incubated in a medium without serum for 24 h (100 μl). After incubation, cell viability was evaluated using the XTT cell proliferation assay kit (Biological Industries) according to the manufacturer's instructions. Briefly, 100 μl of the activation reagent was added to 5 ml of the XTT reagent, followed by 50 μl of activated XTT solution to each well. After 3 h of incubation at 37 °C, color intensity was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader at 450 and 630 nm.
4
Cell Viability Assay with Hybrid Molecules
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The SH-SY5Y and HEK-293 cell lines (2 × 105 cells/mL) were cultured in 96-well tissue microplates (100 µL/well) and allowed to adhere overnight at 37 °C. The conjugate molecules were dissolved in DMEM:nutrient mixture F12 (Ham’s) (1:1) (Biological Industries, Israel) at different concentrations. The negative control was prepared as medium without hybrid molecules and treated in the same manner. 100 µL of medium with or without hybrid molecules were added to each well in triplicate. Following incubation for 24 h at 37 °C, cell viability was evaluated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell proliferation assay kit (Biological Industries, Israel) according to the manufacturer’s instructions. Briefly, 100 µL of activation reagent was added to 5 mL of XTT reagent, followed by the addition of 50 µL of activated-XTT solution to each well. After 2 h incubation at 37 °C, color intensity was measured using an ELISA microplate reader at 450 nm and 630 nm. Results are presented as mean and mean standard error. Each experiment was repeated minimum three times.
5
Cytotoxicity and Hydroxyapatite Formation Assays
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The cytotoxicity assays were carried out in accordance with the guidelines recommended by the American National Standard ISO 10993-5 [58 (link)]. In total, 104 NIH-3T3 cells were first cultured for 1 day and incubated with 20 mg/mL of the materials at 37 °C with 5% CO2 for another day. Dulbecco’s modified Eagle medium was utilized as a control. After incubation, the medium was removed from the wells, and the XTT cell proliferation assay kit (Biological Industries, Kibbutz Beit Haemek, Israel) was used to measure the viability. The absorbances at 492 nm were recorded using an ELISA reader (SPECTROstar Nano, BMG LABTECH, Offenburg, Germany).
For the hydroxyapatite (HA) formation assays, the 80S-ZnAg materials were immersed in PBS for 2 h, 4 h, 8 h, 16 h, 24 h, and 7 days. After immersion, the samples were dried and subjected to XRD and SEM analyses.
For the hydroxyapatite (HA) formation assays, the 80S-ZnAg materials were immersed in PBS for 2 h, 4 h, 8 h, 16 h, 24 h, and 7 days. After immersion, the samples were dried and subjected to XRD and SEM analyses.
6
Cell Viability Assay of Metabolites
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SH-SY5Y cell line (2 × 105 cells/ml) were cultured in 96-well tissue microplates (100 μl per well) and allowed to adhere overnight at 37°C. Metabolites were dissolved at 90°C in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) at various concentrations ranging from 0.2 to 10 mg/ml, followed by gradual cooling of the solution. Each plate was divided, and only half of it was plated with cells. The negative control, represented by zero, was prepared as medium with no metabolites, which was treated in the same manner. Medium (100 μl) with or without metabolites was added to each well. After incubation for 6 hours at 37°C, cell viability was evaluated using the XTT cell proliferation assay kit (Biological Industries) according to the manufacturer’s instructions. Briefly, 100 μl of the activation reagent was added to 5 ml of the XTT reagent, followed by the addition of 100 μl of activated XTT solution to each well. After 2.5 hours of incubation at 37°C, color intensity was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader at 450 and 630 nm. Results are presented as means ± SEM. Each experiment was repeated three times.
7
Cell Proliferation Assay with Compound C
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A cell proliferation assay was performed using a commercial kit, the XTT Cell Proliferation Assay Kit (Biological Industries, Beit Haemek, Israel), as previously described.21 , 22 U251, T98, A172, MCF7, PANC1 and NHA cells were seeded on 96‐well plates at 5.0 × 103 cells (Figure 1 F, Figure 2 B, Figure 4D and Figure 6A) or 1.0 × 104 cells (Figure 1 E, Figure 2 A and Figure 5D) per well. The cells were incubated for 24 hours in an atmosphere of 5% CO2 in air at 37°C in the presence of Compound C, with or without 30 minutes of exposure to an AMF.
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