The S18 or S26 cells were untreated or treated with the compounds to test for 24 h, then harvested and resuspended at 106 cells/mL density in ice-cold DMEM (supplemented with 2% fetal bovine serum). The DNA binding dye hoechst 33342 (Sigma-Aldrich) was added at a final concentration of 5 μg/mL and incubated for 90 min at 37°C in the dark with interval mixing. As a negative control, a subset of the cells were incubated with 5 μM fumitremorgin C (FTC, an inhibitor of ABCG2 that could block the pumping out of hoechst 33342 in CSCs, Merck) for 5 min prior to hoechst 33342 dyeing. After hoechst staining, cells were washed twice then pelleted and maintained at 4°C before FACS analysis. FACS analysis was performed on COULTER EPICS ALTRA™ Flow Cytometer (Beckman Coulter). The hoechst dye was excited with UV laser at 350 nm and its fluorescence was measured at two wavelengths using a 450/40 BP filter (hoechst Blue) and a 675 long pass filter (hoechst Red). Flow cytometry data were analyzed using FlowJo software. At least three independent experiments were performed.
Coulter epics altra flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Coulter Epics Altra flow cytometer is a laboratory instrument designed for high-performance cell analysis and sorting. It utilizes advanced flow cytometry technology to rapidly measure and analyze the physical and fluorescent characteristics of cells or particles suspended in a fluid stream.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Cd90 apc, by BD (1 mentions)
Hla 1 fitc, by BD (1 mentions)
Cd105 pe, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Cd45 apc, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Fitc conjugated anti cd11b monoclonal antibody, by BD (1 mentions)
Annexin 5 fitc binding assay, by BD (1 mentions)
5 protocols using coulter epics altra flow cytometer
1
Hoechst 33342 Staining for CSC Detection
2
Cell Apoptosis and Cell Cycle Analysis
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Kyse 150 and Eca 109 cells of different groups (NC, 25 and 50 nM groups) were collected and adjusted to a concentration of 1x106 cell/ml. Cells then underwent centrifugation at 1,000 x g for 5 min, following which the supernatant was discarded. Samples were then washed twice with cold PBS and centrifuged for a further 5 min at 1,500 x g at 4˚C Cells were resuspended using cooled 70% EtOH and fixed with 70% EtOH overnight at 4˚C. The following day, samples were centrifuged (1,500 x g; 10 min; 4˚C), washed once with PBS, washed twice with normal saline and centrifuged a second time (1,500 x g; 5 min; 4˚C). Cells were then stained with Propidium iodide (50 mg/l; Triton X-100, 1.0%; RNase A, 10 mg/l; Thermo Fisher Scientific, Inc.) at 4˚C in the dark for 30 min. A flow cytometer was used to measure early and late stage cell apoptosis and the cell cycle by flow cytometry (Coulter Epics Altra flow cytometer; Beckman Coulter).
3
Immunophenotypic Characterization of MSCs
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Immunophenotypic characterization of MSCs was performed according to the methodology described by Montesinos et al. [16 (link)]. Monoclonal antibodies against surface markers characteristic of MSCs were used: CD105-PE, CD90-APC, CD73-PE, HLA-I-FITC, HLA-II-PE, and CD45-APC (BD Biosciences, San Diego, CA); CD13-PE and CD14-PE (Caltag, Buckingham, United Kingdom); and CD31-FITC and CD34-APC (Invitrogen, Carlsbad, CA). A total of 1‐1.5 × 106 MSCs were resuspended in 100 mL of phosphate-buffered saline with 3% FBS and 1 mM EDTA (cytometry buffer) and incubated for 20–30 min with the appropriate antibodies. Next, the cells were washed with 1 mL of buffer and fixed with FACS Lysing Solution (BD Biosciences). The samples were analyzed on a Coulter Epics Altra Flow Cytometer (Beckman Coulter, Brea, CA), and at least 10,000 events were collected. The percentages of positive cells and mean fluorescence intensity (MFI) were obtained. The data were analyzed with FlowJo 7.6.1 software.
4
Dual-Marker Flow Cytometry Analysis
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After the surface molecules were stained directly by PE-conjugated anti-Gr-1 monoclonal antibodies and FITC-conjugated anti-CD11b monoclonal antibody (1/100 × for rat monoclonal antibodies, and 1/50 × for mouse monoclonal antibodies; PharMingen, San Diego, CA, USA), the cell surface expression of markers on splenocytes was analyzed by flow cytometry employing a Coulter Epics Altra flow cytometer (Beckman Coulter Co., Miami, FL, USA). Isotype immunoglobulin was used as a control.
5
Annexin V-FITC Apoptosis Assay
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To determine apoptosis in treated cells, an annexin V-FITC binding assay (BD Biosciences Pharmingen, San Diego, CA) was performed following the manufacturer’s guidelines. Treated cells were washed with PBS buffer and then resuspended in the staining solution for 10 minutes before flow cytometry analysis. In brief, 2 × 10^6 cells in a 200 µL suspension were plated in a 6-well plate containing DMEM with 10% FCS. The cells were treated with the extract at IC50 concentrations for 24 hours. Afterward, the cells were harvested, washed, and resuspended in 5 µL of Annexin V solution, followed by incubation in the dark for 10 minutes. Then, 10 µL of propidium iodide (PI) solution was added to the cell suspension before analysis using the Coulter Epics Altra flow cytometer (Beckman Coulter, USA). The acquired data, expressed as a percentage of cells, were analyzed using the BD Accuri™ Flow cytometer. The lower left quadrant (Q3: Annexin-V and PI negative) represented viable cells, the lower right quadrant (Q4: Annexin-V positive) represented early apoptotic cells, the upper right quadrant (Q2: Annexin-V and PI-positive) represented late apoptotic cells, and the upper left quadrant (Q1: PI-positive) represented necrotic cells.
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