The CAMs were synchronized in G2/M phase by treating the cell with nocodazole (100 ng/ml, Abcam) for 12 h [18 (link)]. After soft wash with cold PBS, the CD38+/+ or CD38−/− CAMs was incubated with vehicle (Vehl) or baf for 24 h and then collected by trypsinization. Furthermore, the CAMs was harvested by centrifugation on 700 rpm at 4°C for 10 min and washed twice with PBS. The cells were then fixed in ice-cold 70% ethanol for 30 min at 4℃ and washed twice with PBS. The cells were treated with ribonuclease (100 μg/ml, Abcam) followed by addition of 200 μL propidium iodide (PI, 50 μg/ml, Abcam). Samples were analyzed using a Guava Easycyte Mini Flow Cytometry System and the Guava acquisition and analysis software (Guava Technologies, Hayward, CA). The cell number in G0/G1, S or G2/M phase was divided by the total cell number (G0/G1+S+G2/M) to indicate the percentage of cells in specific phases.
Guava acquisition and analysis software
Manufactured by Merck Group
Sourced in United States
The Guava acquisition and analysis software is a tool designed to facilitate the collection and analysis of data from Guava flow cytometry instruments. The software provides the core functionality to capture and process data generated by Guava flow cytometers, enabling users to perform essential tasks such as data acquisition and analysis.
Automatically generated - may contain errors
5 protocols using guava acquisition and analysis software
1
Cell Cycle Analysis of CD38 CAMs
2
Cell Cycle Analysis of CD38 CAMs
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The CAMs were synchronized in G2/M phase by treating the cell with nocodazole (100 ng/ml, Abcam) for 12 h [18 (link)]. After soft wash with cold PBS, the CD38+/+ or CD38−/− CAMs was incubated with vehicle (Vehl) or baf for 24 h and then collected by trypsinization. Furthermore, the CAMs was harvested by centrifugation on 700 rpm at 4°C for 10 min and washed twice with PBS. The cells were then fixed in ice-cold 70% ethanol for 30 min at 4℃ and washed twice with PBS. The cells were treated with ribonuclease (100 μg/ml, Abcam) followed by addition of 200 μL propidium iodide (PI, 50 μg/ml, Abcam). Samples were analyzed using a Guava Easycyte Mini Flow Cytometry System and the Guava acquisition and analysis software (Guava Technologies, Hayward, CA). The cell number in G0/G1, S or G2/M phase was divided by the total cell number (G0/G1+S+G2/M) to indicate the percentage of cells in specific phases.
3
Measuring Cell Injury via Flow Cytometry
4
Quantifying Autophagy and Lysosomal Activity
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The Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) was used to detect AP 5 (link). Briefly, CAMs (1 × 105/ml) were collected and centrifuged (400 × g, 5 min.) at the end of the treatment. Then, CAMs were incubated with 0.5 ml of freshly diluted Cyto-ID Green Detection Reagent (1:4000) for 30 min. at 37°C in the dark. Without washing, stained CAMs were run in the green (FL1) channel with a Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA, USA) and analysed with Guava acquisition and analysis software (Guava Technologies). The enhancement of Cyto-ID Green dye signal indicates an increase in AP.
In addition, acridine orange (Sigma-Aldrich, St. Louis, MO, USA) was used to detect APLs. CAMs (1 × 106/ml) were stained with acridine orange (1:5000) for 17 min. After washes, CAMs were harvested in phenol red-free growth medium. Green (510–530 nm) and red (>650 nm) fluorescence emission from 104 cells illuminated with blue (488 nm) excitation light was measured with Flow Cytometry System and analysed with Guava acquisition and analysis software. The mean red/green fluorescence ratio was calculated to indicate the change of intracellular APLs.
In addition, acridine orange (Sigma-Aldrich, St. Louis, MO, USA) was used to detect APLs. CAMs (1 × 106/ml) were stained with acridine orange (1:5000) for 17 min. After washes, CAMs were harvested in phenol red-free growth medium. Green (510–530 nm) and red (>650 nm) fluorescence emission from 104 cells illuminated with blue (488 nm) excitation light was measured with Flow Cytometry System and analysed with Guava acquisition and analysis software. The mean red/green fluorescence ratio was calculated to indicate the change of intracellular APLs.
5
Quantifying Autophagy Using Cyto-ID and Acridine Orange
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