O2•− of healthy-MSCs or CKD-MSCs was measured using MitoSOX (Thermo Fisher Scientific, Waltham, MA, USA). The cells in each group were subjected to trypsinization and centrifuged at 600 g for 5 min. The samples were washed and incubated with 10 μM MitoSOX solution in phosphate buffered saline (PBS) at 37 °C for 15 min. Next, the cells were resuspended in 500 μL PBS, and the total number of cells labeled by MitoSOX was measured by fluorescence-activated cell sorting (Sysmex, Kobe, Japan). MitoSOX-positive cells were identified using Flowing Software (DeNovo Software, Los Angeles, CA, USA).
Flowing software
Manufactured by De Novo Software
Sourced in United States
Flowing Software is a comprehensive laboratory data management software. It provides tools for data acquisition, analysis, and visualization. The software supports a wide range of laboratory instruments and can integrate data from multiple sources.
Automatically generated - may contain errors
7 protocols using flowing software
1
Mitochondrial Superoxide Measurement in MSCs
2
Autophagy Quantification in MSCs
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Autophagy staining (Sigma-Aldrich) was used to observe autophagosomes in cultured MSCs. The cells were incubated with autophagy stain (10 μM) for 30 min. After washing with PBS twice, these cells were fixed in 500 μL 4% PFA (paraformaldehyde) for 1 h. These cells were measured for autophagosomes by using FACS (Sysmex, Kobe, Japan). Autophagosome-positive cells were analyzed by using Flowing Software (De Novo Software, Los Angles, CA, USA).
3
Quantifying Mitochondrial Superoxide in hMSCs
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To measure the formation of mitochondrial O2•−, the mitochondrial superoxide of healthy-hMSCs or CKD-hMSCs was measured by means of MitoSOX™ (Thermo Fisher Scientific, Waltham, MA, USA), or TMRE (abcam, Cambridge, UK). These cells were trypsinized for 5 min and then centrifuged at 1200 rpm for 3 min, washed with PBS twice and then incubated with a 10 μM MitoSOX™ solution or 200 nM TMRE solution in PBS at 37 °C for 15 min. After that, the cells were washed more than 2 times with PBS. Next, we resuspended these cells in 500 μL of PBS, and then detected the signals with MitoSOX™ or TMRE by fluorescence-activated cell sorting (FACS; Sysmex, Kobe, Japan). Cell forward scatter levels were determined in MitoSOX™-positive cells, analyzed by means of the Flowing Software (DeNovo Software, Los Angeles, CA, USA).
4
Mitochondrial ROS Detection in MSCs
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To measure the generation of mitochondrial ROS, mitochondrial superoxides of MSCs were indicated using MitoSOX™ (Thermo Fisher). These cells were trypsinized for 5 min and centrifugated at 1200 r/min for 3 min, washed with PBS twice, and then incubated with 10 μM MitoSOX™ solution in PBS at 37 °C for 15 min. Cells were then washed at least twice with PBS. These cells were suspended in 500 μL PBS and MitoSOX™ positive cells were detected using FACS (Sysmex). Cell forward scatter levels indicating MitoSOX™ positive cells were analyzed using Flowing Software (DeNovo Software).
5
Mitochondrial Imaging in Cultured MSCs
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MitoTracker (Sigma-Aldrich, St. Louis, MO, USA) was used to observe mitochondria in cultured MSCs. The cells were incubated with MitoTracker (10 nM) for 15 min. After washing with PBS twice, these cells were suspended in 500 μL PBS, and were used to detect the mitochondrion ratio per cell by using FACS (Sysmex, Kobe, Japan). Cell forward scatter levels indicating MitoSOX™ positive cells were analyzed using Flowing Software (De Novo Software, Los Angles, CA, USA).
6
Mitochondrial Superoxide Generation Assay
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Generation of mitochondrial O2•− in hMSCs was assayed using MitoSOX (Thermo Fisher Scientific). The cells in each group were trypsinized and centrifuged at 600 g for 5 min. The samples were then washed with PBS and incubated with 10 μM MitoSOX solution in phosphate-buffered saline (PBS) at 37 °C for 15 min. Next, the cells were resuspended in 500 μL PBS, and the total number of cells labeled using MitoSOX was measured via FACS (Sysmex, Kobe, Japan). MitoSOX-positive cells (number of events: 104) were identified and analyzed using Flowing Software (DeNovo Software, Los Angeles, CA, USA).
7
Mitochondrial Superoxide Quantification in hMSCs
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To quantify the formation of mitochondrial O2•−, we used TMRE (Abcam, Cambridge, UK) to measure generation of mitochondrial superoxide in hMSCs subjected to oxidative stress. For this, hMSCs were trypsinized for 5 min, centrifuged at 1200 rpm for 3 min, washed with PBS twice, and incubated with 200 nM TMRE solution in PBS at 37 °C for 15 min. The cells were then washed two times with PBS and resuspended in 500 μL PBS. Following this, TMRE signaling was detected by fluorescence-activated cell sorting (FACS; Sysmex, Kobe, Japan). Cellular forward-scatter levels for TMRE-positive cells (number of events: 104 cells) were analyzed using Flowing Software (DeNovo Software, Los Angeles, CA, USA).
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