Antibodies for flow cytometry assays were used for measurement of cell markers, which include FITC anti-mouse/human Ki-67 Antibody (151211, Biolegend, USA), APC anti-human CD8 antibody (980904, Biolegend, USA), PE anti-human CD28 antibody (302907, Biolegend, USA) and PE/Cyanine7 anti-human CD45RO Antibody (304229, Biolegend, USA). Matched immunoglobulin G (IgG) antibodies were used as isotype controls. Flow cytometry assays were performed using according to the manufacturer's instructions. Cell sorting was conducted with a Beckman CytoFLEX S flow cytometer (Beckman) using Becton CytExpert software. Data were analyzed using FlowJo V10 software.
Beckman cytoflex s flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Beckman CytoFLEX S is a flow cytometer designed for cell analysis. It uses light scatter and fluorescent signals to detect and enumerate cells within a sample. The CytoFLEX S can measure multiple parameters simultaneously on individual cells.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex s flow cytometer, by Beckman Coulter (2 mentions)
Apc anti human cd8 antibody, by BioLegend (1 mentions)
Ficoll reagent, by GE Healthcare (1 mentions)
Percoll, by Cytiva (1 mentions)
Beckman cytomics fc500 flow cytometer, by Beckman Coulter (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Apc annexin 5 apoptosis detection kit with pi, by BioLegend (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Stro 1, by R&D Systems (1 mentions)
Annexin binding buffer, by Thermo Fisher Scientific (1 mentions)
Fxcycle pi rnase, by Thermo Fisher Scientific (1 mentions)
Alexa flour 488 annexin 5, by Thermo Fisher Scientific (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit 2, by BD (1 mentions)
Anti cd86 apc, by BD (1 mentions)
Anti cd16 bv421, by BD (1 mentions)
Fixation permeabilization buffer, by BD (1 mentions)
Monensin, by Beyotime (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Anti cd14 fitc, by BD (1 mentions)
12 protocols using beckman cytoflex s flow cytometer
1
Flow Cytometry Assays for Cell Markers
2
Isolation and Phenotyping of Immune Cells
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The ischemic hemisphere was harvested, minced, and suspended in RPMI-1640 media and filtered through a 70 μm cell strainer (12 (link)). Two milliliters of 70% Percoll (Cat#17089019, Cytivia, O’Fallon, MO, United States) were loaded to the bottom of the cell suspension and centrifuged at 600 g for 30 min. Cells at the interphase were collected and re-suspended in phosphate-buffered saline (PBS). For the blood sample, the peripheral mononuclear cells were isolated using the Ficoll reagent (Cat#17-1440-02, GE Healthcare, Sweden). Cells were stained with antibodies against CD45-BV510, CD11b-FITC, CD4-APC-Cy7, and CD8-APC for 30 min at 4°C followed by incubation with 7-AAD for 5 min, washed twice with PBS, and resuspended in 100 μl PBS. All antibodies were purchased from BioLegend Inc., San Diego, CA, United States. The stained cells were analyzed on the Beckman CytoFLEX S flow cytometer (Beckman Coulter, Indianapolis, IN, United States).
3
Examining Cell Cycle Effects of BPQDs@HSA and NK Cells under X-ray
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Flow cytometry was employed to examine the effects of BPQDs@HSA and NK cells under X‐ray radiation on HepG‐2 cell cycle progression. Briefly, BPQDs@HSA (2 µg mL−1) were incubated with NK cells for 12 h and then added to HepG‐2 cells at a ratio of 2.5:1. After 24‐h incubation, the NK cells were removed and washed three times with PBS. Then, the HepG‐2 cells were collected, fixed, and stained with PI in the dark and then analyzed on a Beckman Cytomics FC500 flow cytometer. The collected cells were also stained with Annexin V‐FITC and PI according to the method in manufacturer's instructions of assay kit (Solarbio), and then analyzed on Beckman CytoFLEX S flow cytometer.
4
Mitochondrial Membrane Potential Assay
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HCE cells exposed to hyperosmotic medium for 24 h were collected, and a mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China) was employed to examine the potential of the mitochondrial membrane. Following staining, the stained cells were subjected to analysis using a Beckman Cytoflex S flow cytometer (Beckman, USA).
5
Cell Cycle and Apoptosis Analysis of t-BOOH Treated PC12 Cells
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The flow cytometric analysis was used to determine the cell cycle and apoptosis of PC12 cells induced by t-BOOH and recovered ability of CeO2@ZIF-8. Briefly, PC12 cells were treated with 15 or 20 μM t-BOOH for 2 hours, and then the different concentrations of CeO2@ZIF-8 were added for another 48 hours. Then, the treated cells were collected and fixed with 75% ethanol solution and stained with PI in the dark. The stained cells were analyzed on a Beckman CytoFLEX S flow cytometer.
For analysis of cell apoptosis, PC12 cells were plated in 100-mm dishes with a density of 4 × 104 cells/ml. After cell adherence, the cells were treated with t-BOOH (20 μM) and various concentrations of CeO2@ZIF-8 for 48 hours at 37°C. Then, after removing from the medium and washing with cold PBS, the cells were stained with annexin V and PI according to the experimental method in the assay kit (Solarbio) specification. Cell apoptosis was analyzed using a Beckman CytoFLEX S flow cytometer.
For analysis of cell apoptosis, PC12 cells were plated in 100-mm dishes with a density of 4 × 104 cells/ml. After cell adherence, the cells were treated with t-BOOH (20 μM) and various concentrations of CeO2@ZIF-8 for 48 hours at 37°C. Then, after removing from the medium and washing with cold PBS, the cells were stained with annexin V and PI according to the experimental method in the assay kit (Solarbio) specification. Cell apoptosis was analyzed using a Beckman CytoFLEX S flow cytometer.
6
Apoptosis Assay Using Annexin V-APC/PI
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Apoptotic cell death was detected via Annexin V-APC/ propidium iodide (PI) staining using the apoptosis kit (APC Annexin V Apoptosis Detection Kit with PI, Biolegend, USA). DSCs and hEECs (5 × 105 cells) from the various cultures were trypsinized using 0.25% Trypsin (1 × , Phenol Red; no EDTA; Gibco) for 3 min at 37 °C with 5% CO2 and collected, washed, and resuspended in 100 μL binding buffer included in the apoptosis kit, followed by incubation with 5 μL Annexin V-APC and 10 μL PI at room temperature for 15 min in the dark. Then, 400 μL binding buffer was added and the cell samples were analyzed with a Beckman CytoFLEX S flow cytometer (Beckman Coulter, Inc., USA) using FlowJo software. Annexin V+ PI− cells were in the early stage of apoptosis and Annexin V+ PI+ cells were late apoptotic cells.
7
Annexin V-FITC Apoptosis Assay
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RAW264.7 cells were seeded into 6-well plates at a density of 1 × 106 cells/well and were treated with increasing concentrations of EFBS for 48 h. Cells were detected using the Annexin V-FITC apoptosis detection kit (556547, BD Pharmingen, USA) and analyzed on a Beckman CytoFLEX S flow cytometer (Beckman Coulter, CA.USA).
8
Phenotypic Analysis of DPSCs
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For identification of DPSCs phenotype, 3 × 10E5 DPSCs (passage 3) were incubated with PE- or FITC-conjugated monoclonal antibodies against human CD29, CD44, CD73, CD90, CD105, CD34, CD45 (eBioscience, CA), and Stro-1 (R&D Systems, MN) (sFig. 1 ), and flow cytometric analysis were performed using a Beckman CytoFLEX S flow cytometer (Beckman Coulter, USA).
9
Apoptosis and Cell Cycle Analysis
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To analyze apoptosis, cells were treated as indicated, and allowed to recover for 4 days. Floating cells were collected, and then attached cells were collected with trypsin and combined. After spinning down and washing, cells were incubated with Alexa Flour 488 annexin V and 1 μg/ml propidium iodine in 1x annexin-binding buffer for 15 min in the dark (Thermo). After resuspending in additional binding buffer, cells were analyzed on a CytoFLEX S Flow Cytometer (Beckman) using FL1 and FL3. For cell cycle analysis, 23 h after treatment, cells were pulsed with 20 μM EdU (Thermo), and incubated for an additional hour. Cells were collected with trypsin, washed with 1% BSA in PBS, and then fixed with Click-IT fixative D. After washing with 1% BSA in PBS, the cells were permeabilized with 1x component E for 15 min, before performing Click chemistry with Alexa Flour 488 azide for 30 minutes in the dark. Cells were washed with 1x component E, and then resuspended in 500 μl FxCycle PI/RNase (Thermo) for 15 min before analyzing on Beckman CytoFLEX S Flow Cytometer. Preliminary standard gating for cells versus debris and singlet, and analysis of the results, were conducted with FlowJo™ v10.8 Software (BD Life Sciences).
10
Apoptosis Induction by SHH002-hu1
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5 × 105 MDA-MB-231/MDA-MB-468 cells were seeded in 6-well plates and allowed to adhere. When reaching 70% confluence, cells were incubated with 100 nM SHH002-hu1 for 48 h. Then the cells were stained with Annexin V (AV)-FITC and propidium iodide (PI) following manufacturer’s protocol of FITC Annexin V Apoptosis Detection Kit II (BD Biosciences, USA). Finally, the data of apoptosis were detected by a Beckman CytoFLEX S flow cytometer (Beckman Coulter, USA).
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