After exposure to the different treatments, cells were incubated with a serum-free medium containing CM-H2DCFDA (5 µM) for 30 min at 37 °C with 5% CO2. After removing the CM-H2DCFDA solution, wells were replenished with normal growth medium, and cell cultures were maintained at 37 °C with 5% CO2 for 30 min. Cells were harvested by trypsinization, and fluorescence (λex: 485 nm, λem: 520 nm) was measured using a flow cytometer via the CytoFLEXS benchtop flow cytometer (Beckman Coulter Life Sciences, Brea, CA, USA).
Cytoflexs benchtop flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The CytoFLEXS benchtop flow cytometer is a compact and versatile instrument designed for the analysis of cells and particles. It utilizes flow cytometry technology to provide high-performance data acquisition and analysis capabilities. The CytoFLEXS is capable of simultaneously detecting and measuring multiple parameters of individual cells or particles passing through a laser beam.
Automatically generated - may contain errors
5 protocols using cytoflexs benchtop flow cytometer
1
Intracellular Reactive Oxygen Measurement
2
Ezrin, AKAP95, and Yotiao Knockdown Impacts on BEAS-2B and pHAE Cell Cycle
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BEAS-2B and pHAE cells were grown to 80% confluence and they were transfected using lipofectamine RNAiMax in a 1:1 reagent: siRNA ration in complete growth medium without antibiotics. As described under 2.4, the cells were transfected with 40 nM control siRNA or with a mix of 40 nM Ezrin siRNA, 40 nM AKAP95 siRNA, and 40 nM Yotiao siRNA for 48 h. After transfection, cells were treated with 3 ng/mL TGF-β1 and/or 1% CSE for 24 h. After treatment, cells were trypsinized and fixated with a 95% ethanol PBS solution for 72 h. Total DNA from the cells was stained for 15 min. using a 100 µg/mL propidium iodide PBS solution. After staining, cells were lysed in PBS. Fluorescent excitation of propidium iodide was determined by flow cytometry while using the CytoFLEX S benchtop flow cytometer for at least 10,000 cells (Beckman Coulter Life Sciences, Indianapolis, IN, USA). The quantification of the data was acquired using FlowJoTM (BD, Advancing the World of Health, Ashland, OR, USA).
3
Cell Cycle Analysis with Flow Cytometry
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Cells were seeded, harvested as required and followed by fixing overnight in 70% ethanol at −20°C. Cells were washed in PBS containing 0.5% BSA, before incubation for 30 min at room temperature in PBS containing 0.5% BSA, 5 µg/ml RNase A (ThermoFisher) and 25 µg/ml propidium iodide (Sigma). Samples were analysed by CytoFlex S Benchtop Flow Cytometer (Beckman Coulter). For cell cycle drug treatment, TREX‐BCBL‐1 cells were pre‐treated for 16 h with 10 µM RO‐3306, 0.5 µM nocodazole or 2 mm thymidine before addition of doxycycline. Viral load was measured after 48 h.
4
Multiparametric Flow Cytometry Evaluation
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Flow cytometry analysis using the CytoFLEXS benchtop flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA) was used to evaluate intracellular parameters that are associated with not only oxidative stress but also mitochondrial dysfunction, and cell death. HT-22 cells (40,000 cells/well) seeded in 24-well plates were treated with various conditions. Treatment groups included: control condition (DMSO 1%), erastin 1 µM or 1.5 µM, CE3F4 100 µM, and co-treatment of erastin and CE3F4. Three separate wells were analyzed for each condition and the same number of counts (10,000/well) were measured by flow cytometry. At least three independent experiments were performed, and the Kaluza Analysis 1.5 software was employed for quantification of data.
5
Quantifying Newly Synthesized Proteins
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TREx BCBL1-Rta cells stably expressing scrambled shRNA or shRNAs targeting BUD23 were seeded into a 6-well plate at 1 × 106 cells/well with 2 ml RPMI selection media without methionine which was chased out over 1 h. Cells were treated with Click-iT® L-azidohomoalanine (AHA, 40 µM, Thermo Fischer) for 3 h, then washed in PBS and fixed in PBS containing 4% (v/v) paraformaldehyde for 15 min. Cells were again washed in PBS and permeabilised using PBS containing 0.25% Triton X-100 for 15 min. AHA was stained with a Click-iT® reaction kit (Thermo Fischer) using an alkyne Alexa Fluor™ 488, 1 µM, as described by the manufacturer. Finally, cells were washed in PBS containing 1% BSA and resuspened in PBS containing 0.5% BSA before fluorescence quantification by CytoFlex S Benchtop Flow Cytometer (Beckman Coulter).
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