Cell-cycle analysis was performed using flow cytometry. RMCs were synchronized by incubation in serum-free medium for 24 h and then incubated with the test compounds for 48 h as described above. Then the cells were washed twice with cold phosphate-buffered saline (PBS) and fixed with 75% alcohol for 24 h at 4°C. The fixed cells were collected by centrifugation, washed with PBS, stained for 30 min at room temperature using the Coulter DNA prep reagent kit (Beckman Coulter, Inc., Brea, CA, USA), and finally analyzed using a Beckman Coulter FC 500 (Beckman Coulter, Inc.) together with the CXP software (Beckman Coulter, Inc.).
Dna prep coulter reagent kit
Manufactured by Beckman Coulter
Sourced in France
The DNA Prep Coulter Reagent Kit is a laboratory product designed for the preparation of DNA samples. It provides the necessary reagents and solutions to facilitate the extraction, purification, and concentration of DNA from various biological samples. The kit is intended for use in research and diagnostic applications that require high-quality DNA for further analysis.
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Lab products found in correlation
Gallios flow cytometer, by Beckman Coulter (4 mentions)
Expo32 acquisition software, by Beckman Coulter (2 mentions)
Fc500, by Beckman Coulter (1 mentions)
Cxp software, by Beckman Coulter (1 mentions)
Anti cd3 bv421, by BioLegend (1 mentions)
Staphylococcal enterotoxin b, by Merck Group (1 mentions)
Annexin 5 binding buffer, by Abcam (1 mentions)
Facscalibur, by BD (1 mentions)
7 aad, by Beckman Coulter (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Cell proliferation kit 2 xtt, by Roche (1 mentions)
Annexin 5 fitc pi kit, by Beckman Coulter (1 mentions)
Celltitre blue, by Promega (1 mentions)
Kaluza acquisition software, by Beckman Coulter (1 mentions)
Epics xl flow cytometer, by Beckman Coulter (1 mentions)
Gallios software, by Beckman Coulter (1 mentions)
Epics xl, by Beckman Coulter (1 mentions)
10 protocols using dna prep coulter reagent kit
1
Flow Cytometry-Based Cell Cycle Analysis
2
Staphylococcal Enterotoxin B-Induced T Cell Proliferation
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Peripheral blood mononuclear cells at 2 × 106 viable cells/mL (final concentration) in complete DMEM/F12 were cultured with 5 μg/mL of Staphylococcal enterotoxin B (SEB; Sigma-Aldrich, United Kingdom) for 6 days in 24-well flat-bottom plates at 37°C and 5% CO2. Cells were stained with anti-CD3 BV421 (Biolegend), and anti-CD8 AF488 (Biolegend) for 20 min at 20°C. T cell proliferation was measured using the Coulter DNA Prep Reagent Kit (Beckman Coulter Ltd., High Wycombe, United Kingdom) according to the manufacturer’s instructions. Data were acquired by Flow cytometry (EC800 Sony). Analysis was performed using FlowJo software (FlowJo, United States).
3
Cell Death and Characterization Assessment
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Cell death was evaluated by human FITC-labeled Annexin V (Enzo®) and 7-amino-actinomycin D (7-AAD, Beckman Coulter) staining. Therefore, cells were resuspended in 200 μL Annexin V Binding Buffer (Abcam) with 2 μL Annexin V and 2 μL PI (20 μg/mL), incubated for 15 min, washed and resuspended in PBS/ 5 % FCS prior to analysis. Cells were examined in the FACSCalibur (Becton-Dickinson, Heidelberg, Germany). Cell cycle analysis was performed with the Coulter DNA PREP Reagent kit (Beckman Coulter).
Primary cells were all characterized by staining with a panel of antibodies and flow cytometric analysis (Additional file1 : Table S1). Therefore, we used anti-human EpCAM/TROP1 Phycoerythrin MAb (Clone 158206), anti-human CD31/PECAM-1 APC MAb (Clone 9G11), anti-human VEGF R2/KDR Phycoerythrin MAb (Clone 89106), anti-human CD45 PerCP MAb (Clone 2D1) and anti-human CD14 Fluorescein MAb (Clone 134620, all from R&D systems).
Primary cells were all characterized by staining with a panel of antibodies and flow cytometric analysis (Additional file
4
Chromatography and Spectroscopy Analysis
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Column chromatography: silica gel 60 (70-230 mesh, Sigma-Aldrich). NMR spectra were recorded on a Varian Oxford AV-200 MHz spectrometer, using reference line as a standard. IR spectra were recorded on a Nexus 670 FT-IR instrument from KBr pellets.
The XTT Cell proliferation Kit II was purchased from Roche diagnostics. The Cell Titre-Blue was obtained from Promega. The Coulter® DNA Prep™ Reagent Kit as well as the Annexin V-FITC/PI kit was purchased from Beckman Coulter and the FexiGene DNA Kit from Qiagen. All the chemicals were purchased from Sigma-Aldrich and Merck SA Pty Ltd.
The XTT Cell proliferation Kit II was purchased from Roche diagnostics. The Cell Titre-Blue was obtained from Promega. The Coulter® DNA Prep™ Reagent Kit as well as the Annexin V-FITC/PI kit was purchased from Beckman Coulter and the FexiGene DNA Kit from Qiagen. All the chemicals were purchased from Sigma-Aldrich and Merck SA Pty Ltd.
5
Cell Cycle Analysis by Flow Cytometry
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Cells were detached with trypsin, harvested and washed with 1X PBS (phosphate‐buffered saline), fixed in 70% ethanol, and stored at −20 °C. Before the analysis, fixed cells were centrifuged (1252 g , 5 min) and incubated for 3 min at 37 °C in PBS. After centrifugation, cell pellets were dissociated and incubated with RNAse and propidium iodide using the DNA‐Prep Coulter Reagent kit (Beckman Coulter, Villepinte, France) and were analyzed with a Gallios flow cytometer (Beckman Coulter). A computerized gating was applied on the side and front diffusion to exclude small debris and on a pulse width and an integral red fluorescence peak to remove aggregates. The data were analyzed by the Kaluza® acquisition software (Beckman Coulter).
6
Cell Cycle Analysis by Flow Cytometry
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Adherent and floating cells were pooled, washed with phosphate-buffered saline (PBS 1X) and fixed with ethanol 70 %. Cells were then centrifuged at 2,000 r.p.m. for 5 min and incubated for 30 min at 37 °C in PBS 1X, to allow the release of low-molecular weight DNA. Cell pellets were stained with propidium iodide using the DNA Prep Coulter Reagent Kit (Beckman-Coulter, France). Samples were analysed using Gallios flow cytometer (Beckman Coulter, France).
7
Cell Cycle Analysis by Flow Cytometry
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Cells were detached by trypsinization, washed with PBS, fixed in 70% ethanol and stored at −20 °C until analysis. Fixed cells were centrifuged (2000 rpm, 5 min) and incubated for 30 min at 37 °C in PBS. After centrifugation, cells were resuspended and stained with propidium iodide using the DNA-Prep Coulter Reagent Kit (Beckman Coulter, Villepinte, France) and were analyzed using an EPICS XL flow cytometer (Beckman Coulter). Computerized gating was applied on the side and forward scattering to exclude small debris and on a pulse width and integral peak of red fluorescence to eliminate aggregates. The data were analyzed by Expo32 acquisition software (Beckman Coulter).
8
Cell Cycle Analysis by Flow Cytometry
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Adherent and floating cells were pooled by trypsinisation, washed with phosphate-buffered saline (PBS) and fixed with ethanol 70%. Cells were then centrifuged at 2000 r.p.m. for 5 min and incubated for 30 min at 37°C in PBS, to allow the release of low molecular weight DNA. Cell pellets were stained with propidium iodide using the DNA Prep Coulter Reagent Kit (Beckman Coulter). Samples were analyzed using Gallios flow cytometer (Beckman Coulter) and cell cycle distribution was determined using Gallios software (Beckman-Coulter).
9
Cell Cycle Analysis by Flow Cytometry
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Cells were detached by trypsinization, washed with PBS, fixed in 70% ethanol and stored at −20 °C until analysis. Fixed cells were centrifuged (2000 r.p.m., 5 min) and incubated for 30 min at 37 °C in PBS. After centrifugation, cells were resuspended and stained with propidium iodide using the DNA-Prep Coulter Reagent Kit (Beckman Coulter, Villepinte, France) and were analysed using an EPICS XL flow cytometer (Beckman Coulter). Computerized gating was applied on the side and forward scatter to exclude small debris and on a pulse width and integral peak of red fluorescence to eliminate aggregates. The data were analysed by Expo32 acquisition software (Beckman Coulter).
10
Cell Cycle Analysis by Flow Cytometry
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Adherent and floating cells were pooled, washed with phosphate-buffered saline (PBS 1X) and fixed with ethanol 70%. Cells were then centrifuged at 2000 rpm for 5 min and incubated for 30 min at 37°C in PBS 1X, to allow the release of low-molecular weight DNA. Cell pellets were stained with propidium iodide using the DNA Prep Coulter Reagent Kit (Beckman-Coulter). Samples were analyzed with a Gallios flow cytometer (Beckman Coulter).
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