(1) Cellular glucose uptake was determined by flow cytometry analysis of uptake of 2-[N-(7-nitrobenz-2-oxa-1, 3-diazo-l-4-yl) amino]-2-deoxy-d -glucose (2-NBDG). Breast cancer cells were incubated with 50 μmol/L of 2-NBDG for 30 min at 37 °C in a humidified atmosphere containing 5% CO2/95% air. The fluorescence intensity of 2-NBDG taken up by the cells was analyzed on an EasyCyte Plus Flow Cytometry System (Millipore, Chicago, IL, USA). (2) The glucose concentration in the medium was measured using an Amplex Red Glucose/Glucose oxidase assay kit (Invitrogen). The absorbance of the samples was measured using a Varioskan multimode microplate spectrophotometer (Thermo, MA) and used to calculate the concentration of glucose in the medium.
Easycyte plus flow cytometry system
Manufactured by Merck Group
Sourced in United States
The EasyCyte Plus Flow Cytometry System is a compact and automated flow cytometry instrument designed for routine cell analysis. The system utilizes laser-based technology to detect and measure various properties of cells or particles suspended in a fluid stream.
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6 protocols using easycyte plus flow cytometry system
1
Glucose Uptake and Concentration Measurement
2
Cellular Glucose Uptake Quantification
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(1) Cellular glucose uptake was determined by ow cytometry analysis of uptake of 2-[N-(7-nitrobenz-2oxa-1, 3-diazo-l-4-yl) amino] -2-deoxy-D-glucose (2-NBDG). Breast cancer cells were incubated with 50μmol/L of 2-NBDG for 30 min at 37 °C in a humidi ed atmosphere containing 5% CO 2 /95% air. The uorescence intensity of 2-NBDG taken up by the cells was analyzed on an EasyCyte Plus Flow Cytometry System (Millipore, Chicago, IL, USA). (2) The glucose concentration in the medium was measured using an Amplex Red Glucose/Glucose oxidase assay kit (Invitrogen). The absorbance of the samples was measured using a Varioskan multimode microplate spectrophotometer (Thermo, MA) and used to calculate the concentration of glucose in the medium.
3
Cellular Glucose Uptake Quantification
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(1) Cellular glucose uptake was determined by ow cytometry analysis of uptake of 2-[N-(7-nitrobenz-2oxa-1, 3-diazo-l-4-yl) amino] -2-deoxy-D-glucose (2-NBDG). Breast cancer cells were incubated with 50μmol/L of 2-NBDG for 30 min at 37 °C in a humidi ed atmosphere containing 5% CO 2 /95% air. The uorescence intensity of 2-NBDG taken up by the cells was analyzed on an EasyCyte Plus Flow Cytometry System (Millipore, Chicago, IL, USA). (2) The glucose concentration in the medium was measured using an Amplex Red Glucose/Glucose oxidase assay kit (Invitrogen). The absorbance of the samples was measured using a Varioskan multimode microplate spectrophotometer (Thermo, MA) and used to calculate the concentration of glucose in the medium.
4
Cellular Glucose Uptake Quantification
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(1) Cellular glucose uptake was determined by ow cytometry analysis of uptake of 2-[N-(7-nitrobenz-2oxa-1, 3-diazo-l-4-yl) amino] -2-deoxy-D-glucose (2-NBDG). Breast cancer cells were incubated with 50μmol/L of 2-NBDG for 30 min at 37 °C in a humidi ed atmosphere containing 5% CO 2 /95% air. The uorescence intensity of 2-NBDG taken up by the cells was analyzed on an EasyCyte Plus Flow Cytometry System (Millipore, Chicago, IL, USA). (2) The glucose concentration in the medium was measured using an Amplex Red Glucose/Glucose oxidase assay kit (Invitrogen). The absorbance of the samples was measured using a Varioskan multimode microplate spectrophotometer (Thermo, MA) and used to calculate the concentration of glucose in the medium.
5
Measuring Mitochondrial Membrane Potential
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The mitochondrial membrane potential (Δψm) was measured by flow cytometer using the cationic lipophilic green fluorochrome rhodamine 123 (19 (link)). MCF-7 cells were treated with various concentrations of THC for 24 h and the treated cells were harvested, washed twice with PBS, incubated with 1 mM rhodamine 123 at 37°C for 30 min, and washed twice with PBS. Fluorescence was determined by a Guava EasyCyte Plus Flow Cytometry System.
6
Apoptosis Determination in A549 Cells
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Amount of Annexin V positive cells was determined using PE Annexin V Apoptosis Detection Kit (BD Biosciences) according to manufacture’s recommendation. Briefly, A549 cells were treated with 300 μM AOAA, 300 nM CPT or combination of both for 1, 3, 9 and 24 h. Number of Annexin V positive cells was determined using EasyCyte™ Plus Flow Cytometry System (Guava).
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