Cytation 5 luminescence microplate reader, by Agilent Technologies - Product details

1

Cell Proliferation Quantification Using WST-1 Assay

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Cell proliferation was quantified using the WST-1 colorimetric assay (#05015944001; Sigma) according to the manufacturer’s instructions. Briefly, on day 1, B16-OVA and B16-OVA-hCD19 cells were seeded in a 96-well plate (10³ cells per well) in 200 μl of DMEM medium supplemented with 10% FBS, 100 unit/ml penicillin and 100 μg/mL streptomycin. After incubation for the indicated times, 20 μl of WST-1 was added to each well and incubated at 37°C, 5% CO₂ for 2 h. The cell plates were shaken for 1 min on a shaker. The absorbance of each well against a background control (medium with WST-1 only) was measured at 450 nm using a Cytation 5 luminescence microplate reader (BioTek). Data plots were generated by using Prism version 8.0.0 software (GraphPad).

Nguyen N.T., Huang K., Zeng H., Jing J., Wang R., Fang S., Chen J., Liu X., Huang Z., You M.J., Rao A., Huang Y., Han G, & Zhou Y. (2021). Nano-Optogenetic Engineering of CAR T-cells for Precision Immunotherapy with Enhanced Safety. Nature nanotechnology, 16(12), 1424-1434.

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Photostimulation of Engineered T Cells

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Jurkat T cells expressing conventional CARs, LiCAR, or defective LiCAR (10⁵ cells/well) were co-cultured with cognate CD19-positive Raji cells or non-cognate CD19-negative K562 cells at indicated effector T cell: target cell (E/T) ratios or at the ratio of 1:3 in a 96-well flat-bottom microplate (#E17073EF; Greiner Bio-one, Monroe, North Carolina, USA). Plates with cells were incubated at 37 °C in a humidified atmosphere under 5% CO₂ and either kept in the dark or subjected to photostimulation (470 nm at a power density of 40 mW/cm²) for 1 to 25 minutes and then with pulsed blue light (10-30 sec ON, 100 sec OFF) for up to 8 hours. Cell pellets were then harvested, and luciferase activity was assayed by using the Bright-Glo Luciferase Assay System (Promega, Fitchburg, Wisconsin, USA) on the Cytation 5 luminescence microplate reader (BioTek, Winooski, Vermont, USA). Data plots were generated by using the Prism version 8.0.0 software (GraphPad, San Diego, California, USA).

Nguyen N.T., Huang K., Zeng H., Jing J., Wang R., Fang S., Chen J., Liu X., Huang Z., You M.J., Rao A., Huang Y., Han G, & Zhou Y. (2021). Nano-Optogenetic Engineering of CAR T-cells for Precision Immunotherapy with Enhanced Safety. Nature nanotechnology, 16(12), 1424-1434.

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Photostimulation of Engineered T Cells

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Jurkat T cells expressing conventional CARs, LiCAR, or defective LiCAR (10⁵ cells/well) were co-cultured with cognate CD19-positive Raji cells or non-cognate CD19-negative K562 cells at indicated effector T cell: target cell (E/T) ratios or at the ratio of 1:3 in a 96-well flat-bottom microplate (#E17073EF; Greiner Bio-one, Monroe, North Carolina, USA). Plates with cells were incubated at 37 °C in a humidified atmosphere under 5% CO₂ and either kept in the dark or subjected to photostimulation (470 nm at a power density of 40 mW/cm²) for 1 to 25 minutes and then with pulsed blue light (10-30 sec ON, 100 sec OFF) for up to 8 hours. Cell pellets were then harvested, and luciferase activity was assayed by using the Bright-Glo Luciferase Assay System (Promega, Fitchburg, Wisconsin, USA) on the Cytation 5 luminescence microplate reader (BioTek, Winooski, Vermont, USA). Data plots were generated by using the Prism version 8.0.0 software (GraphPad, San Diego, California, USA).

Nguyen N.T., Huang K., Zeng H., Jing J., Wang R., Fang S., Chen J., Liu X., Huang Z., You M.J., Rao A., Huang Y., Han G, & Zhou Y. (2021). Nano-Optogenetic Engineering of CAR T-cells for Precision Immunotherapy with Enhanced Safety. Nature nanotechnology, 16(12), 1424-1434.

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4

Cell Proliferation Quantification Using WST-1 Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell proliferation was quantified using the WST-1 colorimetric assay (#05015944001; Sigma) according to the manufacturer’s instructions. Briefly, on day 1, B16-OVA and B16-OVA-hCD19 cells were seeded in a 96-well plate (10³ cells per well) in 200 μl of DMEM medium supplemented with 10% FBS, 100 unit/ml penicillin and 100 μg/mL streptomycin. After incubation for the indicated times, 20 μl of WST-1 was added to each well and incubated at 37°C, 5% CO₂ for 2 h. The cell plates were shaken for 1 min on a shaker. The absorbance of each well against a background control (medium with WST-1 only) was measured at 450 nm using a Cytation 5 luminescence microplate reader (BioTek). Data plots were generated by using Prism version 8.0.0 software (GraphPad).

Nguyen N.T., Huang K., Zeng H., Jing J., Wang R., Fang S., Chen J., Liu X., Huang Z., You M.J., Rao A., Huang Y., Han G, & Zhou Y. (2021). Nano-Optogenetic Engineering of CAR T-cells for Precision Immunotherapy with Enhanced Safety. Nature nanotechnology, 16(12), 1424-1434.

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Cytation 5 luminescence microplate reader

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4 protocols using cytation 5 luminescence microplate reader

Cell Proliferation Quantification Using WST-1 Assay

Photostimulation of Engineered T Cells

Photostimulation of Engineered T Cells

Cell Proliferation Quantification Using WST-1 Assay

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