The apoptotic response after genotoxic drug treatment was measured using flow cytometry for sub-G1 determination. Supernatant and attached cells were collected, washed once with PBS and fixed in 70% ethanol. Ethanol-fixed cells were stained with propidium iodide (PI) at room temperature for 1 h in PBS containing 20 μg/ml PI (Sigma–Aldrich), 200 μg/ml RNase A, and 0.1% Triton X-100. The percentage of sub-G1 cells was calculated using the CytoSoft software (Millipore, Billerica, MA, USA). For γH2AX and active caspase-3 immunostaining, cells were fixed with 1% formaldehyde and then with 70% ethanol. Afterwards, the cells were blocked, permeabilized, incubated with either primary mouse monoclonal antibody to γH2AX (Ser-139) (Upstate Biotechnology, Lake Placid, NY, USA) and diluted 1:500, or mouse anti-active caspase 3 (BD, Pharmigen, San Diego, CA, USA) diluted 1:50 for 2 h at room temperature. This was followed by incubation with anti-mouse FITC secondary antibody (Sigma-Aldrich) that was diluted 1:200 for 1 h at room temperature. The percentage of γH2AX positive cells was again calculated using the CytoSoft software (Millipore).
Cytosoft software
Manufactured by Merck Group
Sourced in United States, Germany
CytoSoft is a laboratory software application designed to facilitate the analysis and management of cell culture data. The software provides tools for capturing, organizing, and visualizing data generated from various cell-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
Guava cell cycle reagent, by Merck Group (3 mentions)
Guava nexin kit, by Merck Group (2 mentions)
Guava nexin reagent, by Merck Group (2 mentions)
Guava easycyte system, by Merck Group (1 mentions)
Anti ve cadherin antibody, by Abcam (1 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Guava pca instrument, by Merck Group (1 mentions)
Guava personal cell analysis flow cytometer, by Merck Group (1 mentions)
Annexin 5 pe apoptosis detection kit, by Merck Group (1 mentions)
Flowcellect mitodamage kit, by Merck Group (1 mentions)
12 well plates, by AGC Techno Glass (1 mentions)
Guava easycyte mini, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate, by Merck Group (1 mentions)
Mouse anti active caspase 3, by BD (1 mentions)
Mtt reagent, by Promega (1 mentions)
Guava system, by Merck Group (1 mentions)
Guava easycyte plus, by Merck Group (1 mentions)
Stempro kits, by Thermo Fisher Scientific (1 mentions)
Igg1 fitc, by Thermo Fisher Scientific (1 mentions)
Igg2b fitc, by Thermo Fisher Scientific (1 mentions)
16 protocols using cytosoft software
1
Quantifying Apoptosis and DNA Damage
2
Endothelial Differentiation of ASCs
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ASCs were incubated in EDM for the indicated days. CD31 and vascular endothelial (VE)-cadherin are endothelial cell markers. The proportion of CD31 or VE-cadherin positive cells was determined using immunocytochemistry and flow cytometry analysis. Briefly, the cells were harvested and stained with anti-CD31 antibody (Abcam) at 1:100 in 3% BSA/PBS, or anti-VE-cadherin antibody (Abcam) at 1:50 in 3% BSA/PBS. The cells were incubated in the primary antibody at room temperature overnight at 4°C in darkness. The cells were washed twice with ice-cold PBS by centrifugation at 400g for 5 minutes and then incubated in AF488-labeled anti-mouse secondary antibody (Thermo Fisher Scientific) diluted in 3% BSA/PBS at room temperature for 30 minutes in darkness. The cells were washed twice with PBS and subjected to flow cytometry analysis. Flow cytometry analysis was performed using a Guava EasyCyte System equipped with CytoSoft software (EMD Millipore, Billerica, MA, http://www.emdmillipore.com ). The ExpressPlus program (De Novo Software, Glendale, CA, https://www.denovosoftware.com ) was selected to perform the analysis.
3
Cell Cycle Analysis of PRFR Treatment
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HepG2 cells were grown in 6-well plates (2 × 105 cells/well) for 24 h and incubated with or without various concentrations of PRFR (0–40 µg/mL) for 48 h. The cell suspension was then prepared on ice and stained with propidium iodide (Guava Cell Cycle Reagent) for 30 min according to the Guava Cell Cycle Assay protocol. Cell cycle phase distribution was performed on a Guava PCA Instrument using CytoSoft Software (Merck-Millipore, Darmatadt, Germany).
4
HAMLET-Induced Apoptosis Analysis
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We used the Flow Cellect Mito Damage Kit and Annexin V-PE Apoptosis Detection Kit obtained from EMD Millipore in the United States. To perform the flow cytometric analysis and understand how cells responded to HAMLET, we seeded 100,000 to 130,000 cells per well. After one day, we treated these cells with different concentrations of HAMLET for 6 h. We then detached the cells, stained them with Annexin V-PE and 7-AAD, and analyzed them using the Guava Personal Cell Analysis Flow Cytometer and CytoSoft software (version 2.1.4; Guava; EMD Millipore, Burlington, MA, USA).
5
Gilteritinib's Impact on Cell Cycle in MV4-11 Cells
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MV4-11 cells were seeded in 12-well plates (AGC Techno Glass Co. Ltd., Shizuoka, Japan) at a concentration of 2 × 105 cells/well and cultured overnight. The cells were treated with gilteritinib concentrations of 1, 3, 10, and 30 nM or vehicle (0 nM), and incubated for 24 hours.
The cells were subsequently harvested and fixed in ice-cold 70% ethanol and maintained at 4°C. Following fixation, the cells were washed with phosphate-buffered saline (PBS) and were resuspended in Guava® Cell Cycle Reagent (Merck Millipore Corporation, Darmstadt, Germany). Cell cycle distribution was measured using a Guava® PCA microcytometer (Merck Millipore Corporation), and was analyzed in 5000 cells per sample using CytoSoft™ software (Merck Millipore Corporation). The mean percentages of cells in sub-G1, G1, S, and G2/M phases were derived from four independent assays.
The cells were subsequently harvested and fixed in ice-cold 70% ethanol and maintained at 4°C. Following fixation, the cells were washed with phosphate-buffered saline (PBS) and were resuspended in Guava® Cell Cycle Reagent (Merck Millipore Corporation, Darmstadt, Germany). Cell cycle distribution was measured using a Guava® PCA microcytometer (Merck Millipore Corporation), and was analyzed in 5000 cells per sample using CytoSoft™ software (Merck Millipore Corporation). The mean percentages of cells in sub-G1, G1, S, and G2/M phases were derived from four independent assays.
6
Measuring Cellular Oxidative Stress
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Cells were incubated with 20 μM 2′,7′-dichlorofluorescin diacetate (Sigma) in the culture medium for 20 min, detached with trypsin, and collected in 1 mL of PBS. Cells were washed two times with 500 μL of PBS and analyzed on a Guava EasyCyte mini instrument using Cytosoft software version 4.2.1 (Merck Millipore).
7
Purification of Spermatocytes and Spermatids
8
Cytotoxicity and apoptosis assays
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Cells (1x104) seeded in a 96-well plate for overnight were treated with HCQ, RAD001, bafilomycin A1 (50 nM), or spautin-1 (10 μM) for two days. MTT reagent (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, Promega) was added for an hour before spectrophotometric measurement at 490 nm. Each measurement was made in triplicate, and the average value was calculated. The assay was performed on three biological replicates.
For apoptosis assays, cells were seeded overnight and treated with HCQ and RAD001 as described above. Cells were then harvested and stained with Annexin V-PE and 7-AAD (PM2) using a Guava Nexin kit (Millipore). Non-apoptotic (annexin V- and 7-AAD-), early apoptotic (annexin V+ and 7-AAD-), and late apoptotic (annexin V+ and 7-AAD+) cells were measured by Guava System (Millipore), and the measurement was analyzed by CytoSoft software (Millipore).
For apoptosis assays, cells were seeded overnight and treated with HCQ and RAD001 as described above. Cells were then harvested and stained with Annexin V-PE and 7-AAD (PM2) using a Guava Nexin kit (Millipore). Non-apoptotic (annexin V- and 7-AAD-), early apoptotic (annexin V+ and 7-AAD-), and late apoptotic (annexin V+ and 7-AAD+) cells were measured by Guava System (Millipore), and the measurement was analyzed by CytoSoft software (Millipore).
9
Porcine Stem Cell Characterization
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Adipogenesis, chondrogenesis, and osteogenesis were tested with StemPro® kits (Gibco) which were used according to the manufacturer’s instructions (Additional file 1 ).
The immunophenotype of porcine WJCs was analyzed using mouse monoclonal antibodies to porcine CD31 (LCI-4):IgG1-RPE, CD45 (K252.1E4):IgG1-FITC, and SLA class II DR (2E9/13):IgG2b-FITC (AbD Serotec, Bio-Rad, Hercules, CA, USA), porcine CD105 (MEM-229):IgG2a-FITC and CD90 (5E10):IgG1-FITC (Abcam, Cambridge, MA, USA), and porcine CD44 (MEM-263):IgG1-FITC (Thermo Scientific, Middletown, VA, USA). All isotype control antibodies were derived from mice (IgG1-FITC, IgG1-RPE, IgG2a-FITC, and IgG2b-FITC) and were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies for porcine CD73 were not available and monoclonal antibodies against human CD73 do not cross-react with porcine CD73 [10 (link), 11 (link)]. Cell suspensions (1 × 107 cells/ml in 100 μL PBS) were incubated with antibodies or isotype controls (final concentration 3 μg/mL) for 45 min at 4 °C in the dark. Fluorescence was analyzed with a microcapillary cytometer (Guava EasyCyte Plus and Cytosoft™ software, Millipore).
The immunophenotype of porcine WJCs was analyzed using mouse monoclonal antibodies to porcine CD31 (LCI-4):IgG1-RPE, CD45 (K252.1E4):IgG1-FITC, and SLA class II DR (2E9/13):IgG2b-FITC (AbD Serotec, Bio-Rad, Hercules, CA, USA), porcine CD105 (MEM-229):IgG2a-FITC and CD90 (5E10):IgG1-FITC (Abcam, Cambridge, MA, USA), and porcine CD44 (MEM-263):IgG1-FITC (Thermo Scientific, Middletown, VA, USA). All isotype control antibodies were derived from mice (IgG1-FITC, IgG1-RPE, IgG2a-FITC, and IgG2b-FITC) and were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies for porcine CD73 were not available and monoclonal antibodies against human CD73 do not cross-react with porcine CD73 [10 (link), 11 (link)]. Cell suspensions (1 × 107 cells/ml in 100 μL PBS) were incubated with antibodies or isotype controls (final concentration 3 μg/mL) for 45 min at 4 °C in the dark. Fluorescence was analyzed with a microcapillary cytometer (Guava EasyCyte Plus and Cytosoft™ software, Millipore).
10
Quantifying Apoptosis and DNA Damage
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The apoptotic response after genotoxic drug treatment was measured using the flow cytometric method of sub-G1 determination. Supernatant and attached cells were collected, washed once with PBS and fixed in 70% ethanol. Ethanol-fixed cells were stained with propidium iodide (PI) at room temperature for 1 h in PBS containing 20 μg/ml PI (Sigma-Aldrich, St. Louis, MO, USA), 200 μg/ml RNase A and 0.1% Triton X-100. The percentage of sub-G1 cells was calculated using the CytoSoft software (Millipore, Darmstadt, Germany). For γH2AX immunostaining, cells were fixed with 1% formaldehyde and then with 70% ethanol. Afterwards, the cells were blocked, permeabilized and incubated with primary mouse monoclonal antibody to γH2AX (Ser-139) at 1 : 1000 (Upstate Biotechnology, Upstate, NY, USA) and diluted 1 : 500 for 1 h at room temperature, followed by incubation with anti-mouse FITC secondary antibody (Sigma-Aldrich) that was diluted 1 : 200 for 1 h at room temperature.
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