Cellular apoptosis was performed using an Annexin V-FITC/PI Cell apoptosis assay kit. In brief, the HL-7702 cells were treated with T-2 toxin at final concentrations of 0, 0.1, 1.0, and 10 μg/L for 24 h. The HL-7702 cells in control group were incubated with the vehicle, i.e., 0.1% DMSO in DMEM. After centrifugation and resuspension in staining buffer, the cells were incubated in Annexin V-FITC and PI solution for 20 min at room temperature. Fluorescence signal was immediately detected by flow cytometry (Beckman Coulter, Gallios, Indianapolis, IN, USA), and data were analyzed using CXP analysis software version 2.2 (Beckman Coulter Inc., Brea, CA, USA).
Cxp analysis software version 2
Manufactured by Beckman Coulter
Sourced in United States
CXP analysis software version 2.2 is a data analysis tool designed for use with Beckman Coulter flow cytometry instruments. The software provides advanced data processing and visualization capabilities to support the analysis of cell populations and fluorescent marker expression.
Automatically generated - may contain errors
Lab products found in correlation
Fc500 flow cytometer, by Beckman Coulter (2 mentions)
Cytomics fc500, by Beckman Coulter (2 mentions)
Propidium iodide, by Merck Group (1 mentions)
Fc500 flow cytometry, by Beckman Coulter (1 mentions)
Cytomics fc500 cytometer, by Beckman Coulter (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Propidium iodine, by Merck Group (1 mentions)
Cd34 pe, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Mxp software, by Beckman Coulter (1 mentions)
Cd166 pe, by R&D Systems (1 mentions)
Cd105 fitc, by R&D Systems (1 mentions)
8 protocols using cxp analysis software version 2
1
HL-7702 Cell Apoptosis Assay
2
Cell Cycle Analysis by Flow Cytometry
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Cells were treated with 10 µM rotenone, rotenoisin B, and control DMSO (0.1%) for 48 h and then cells harvested by trypsinization and washed with PBS. Cells (1 × 106) were resuspended in 70% Ethanol (kept at −20 °C) 5 mL drop wise manner then cells were stored at 4 °C for overnight for fixation. Cells were washed with PBS and suspended staining solution containing 0.5% Triton X-100, 10 µg/mL RNAse-A, 20 µg/mL PI, 1 mM EDTA and incubated at room temperature for 20 to 30 min in dark. Cells were then analyzed for DNA content profile by flow cytometry FC500 flow cytometer (Beckman-Coulter, Fullerton, CA, USA) from 10,000 events per sample. Data from flow cytometry was analyzed using CXP analysis software version 2.2 (Beckman-Coulter, Fullerton, CA, USA).
3
Cell Cycle Analysis by Flow Cytometry
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MCF7 cells were obtained and analyzed as previously described [44 (link)]. Briefly, DNA was stained in ethanol-fixed cells with propidium iodide (50 µg/mL; Sigma-Aldrich, Lyon, France) and Ki-67 protein was eventually detected using a FITC-coupled mouse anti-human Ki-67 antibody (Becton-Dickinson, Le pont de Claix, France). Cell fluorescence was measured by flow cytometry (Cytomics FC500; Beckman Coulter, Villepinte, France). Cell-cycle distribution was analyzed for 20,000 events using MultiCycle AV Software, Windows version (Phoenix Flow System, San Diego, CA, USA) to obtain histograms of cell repartition in each cell-cycle phase and CXP Analysis software version 2.2 (Beckman Coulter, Villepinte, France) to obtain dot-plots of DNA/Ki-67 double-staining.
4
P-glycoprotein Expression Analysis
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Cells were seeded in a 6-cm flat-bottom tissue culture plate (Sarstedt) at a density of 200,000 cells per plate in 3 mL drug-free medium. The plate was incubated in a humidified incubator at 37 °C with 5% CO2 for 24 h. After 24 h, the spent medium was discarded. The adherent cells were then washed twice with PBS and trypsinized. Cells were collected from the culture vessel in 5 mL PBS, and pelleted by centrifugation at 500 × g for 5 min. The cell pellet was subsequently resuspended in 80 µL stain buffer (2% FBS in PBS) and either 20 µL R-phycoerythrin-conjugated mouse anti-human P-gp antibody (Clone 17F9, BD Pharmingen, BD Biosciences) or 20 µL R-phycoerythrin-conjugated mouse IgG2b κ isotype control antibody (Clone 27–35, BD Pharmingen, BD Biosciences). After a 30-min incubation in the dark at room temperature, 1 mL PBS was added to each cell-antibody mixture. The cells were then pelleted by centrifugation at 500 × g for 5 min, resuspended in 500 μL PBS, and loaded onto an FC500 flow cytometer (Beckman Coulter). Samples were analysed under channel FL3 using an excitation wavelength of 488 nm and an emission wavelength of 585 ± 15 nm. Analyses were stopped at 20,000 events or after 300 s. The results were captured and analysed using the CXP Analysis software version 2.2 (Beckman Coulter), with all samples being ungated.
5
Cell Cycle Analysis by Flow Cytometry
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Subconfluent cells were treated as indicated and labeled with propidium iodide [42] (link). FITC-coupled mouse anti-human Ki-67 antibody or irrelevant isotype antibody for control (BD Pharmingen, France) was eventually added before DNA staining with propidium iodide. Cell cycle distribution based on 2n or 4n DNA content was analyzed by flow cytometry (Cytomics FC500; Beckman Coulter, France) and 20,000 events were analyzed with MultiCycle AV Software, Windows version (Phoenix Flow System; San Diego, CA, USA) and CXP Analysis software version 2.2 (Beckman Coulter).
6
Intracellular ROS Quantification by DCFH-DA
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Intracellular ROS was detected using fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). In brief, Huh-7 cells in 6-well plate were incubated with 10 µM rotenone and rotenoisin B and DMSO control for 4 h. After treatment of rotenoisin B, 3 mM N-acetyl-l -cysteine (NAC) was added. Cells were then trypsinized and washed in PBS and incubated with 20 µM H2DCFDA at 37 °C for 30 min in the dark. Further cells were washed with PBS and DCF fluorescence was detected using flow cytometry FC500 (Beckman-Coulter, Fullerton, CA, USA). ROS were expressed as mean fluorescence intensity (MFI), which was CXP analysis software version 2.2 (Beckman-Coulter, Fullerton, CA, USA).
7
Cell Cycle Analysis of MG-63 Cells
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MG-63 cells were plated on 12-well plates at a density of 1×104 cells/well, treated with different concentrations of DG, and then incubated for 24 h. Adherent cells were harvested with trypsin (Gibco), washed in phosphate-buffered saline (PBS), collected by centrifugation at 400 × g for 10 min, and then fixed with 75% ethanol at 4°C for 24 h. Cell pellets were then collected by centrifugation at 400 × g for 10 min and incubated with propidium iodine (PI; Sigma-Aldrich) solution (0.1% Triton X-100, 0.2 µg/ml Ribonuclease A and 40 µg/ml PI) for 30 min. Cell cycle phase was determined by flow cytometry using a Cytomics FC500 cytometer (Beckman Coulter, Brea, CA, USA) and CXP Analysis Software version 2.1 (Beckman Coulter). Other materials and reagents not specified were obtained from Sigma-Aldrich or Merck Millipore (Billerica, MA, USA).
8
Phenotypic Characterization of Mesenchymal Stem Cells
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For phenotypic characterization of MSCs, monoclonal antibodies conjugated with different fluorochromes (Fluorescein [FITC]/Phycoerythrin [PE]/Alexa-647 [AL-647]), which combine a number of both positive and negative MSC membrane markers, were used. Positive markers used were CD105 FITC (R&D Systems); CD90 AL-647 (AbDSerotec,); HLA Class I FITC (Cytognos); CD73 PE (BD Bioscience) and CD166 PE (R&D Systems). Negative markers used were CD34 PE (BD Bioscience); HLA class IIPE (Cytognos); CD80 AL-647 (AbDSerotec); CD45 FITC (Cytognos) and CD31 FITC (Cytognos). Furthermore, suitable isotopic controls for FITC, PE (Cytognos) and AL-647 (AbDSerotec) were used as controls for specificity of the monoclonal antibodies.
The labelled cells were acquired with a flow cytometer FC500 MPL Cytomics (Beckman Coulter) using the MXP software (Beckman Coulter). Nonviable cells were discarded using the labeling reagent LIVE&DEAD (Invitrogen), and the collected data were analyzed with the CXP analysis software, version 2.1 (Beckman Coulter). Criteria for the administration of MSCs in our present clinical trial included a viability >95%, absence of microbial contamination (bacteria, fungus, virus or mycoplasma), expression of CD105, CD90, HLA I, CD73 and CD166 for >90% of cells and absence of CD34, CD80, HLA II, CD45 and CD31 (expression of each <5%), as assessed using flow cytometry (Figure 1).
The labelled cells were acquired with a flow cytometer FC500 MPL Cytomics (Beckman Coulter) using the MXP software (Beckman Coulter). Nonviable cells were discarded using the labeling reagent LIVE&DEAD (Invitrogen), and the collected data were analyzed with the CXP analysis software, version 2.1 (Beckman Coulter). Criteria for the administration of MSCs in our present clinical trial included a viability >95%, absence of microbial contamination (bacteria, fungus, virus or mycoplasma), expression of CD105, CD90, HLA I, CD73 and CD166 for >90% of cells and absence of CD34, CD80, HLA II, CD45 and CD31 (expression of each <5%), as assessed using flow cytometry (Figure 1).
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