Cultured cells were removed from flasks using cell dissociation buffer (Thermofisher, Waltham, MA) and resuspended in ice cold flow cytometry buffer (Dulbecco PBS, 1% BSA, 0.1% NaN3, 5 mM EDTA) at 100 μl/106 cells. Next, 50 μM peptides or 3 μg/ml primary mouse mAbs diluted in flow cytometry buffer were incubated with cells at 4°C with gentle shaking for 1 h. After washing three times with flow cytometry buffer to remove unbound peptides or antibodies, cells were incubated with 0.5 μg/ml APC-conjugated streptavidin or secondary antibodies (Thermofisher, Waltham, MA) at 4°C for 45 min. After washing three times, cells were kept at 4°C until analysis using a BD CyAn ADP flow cytometer (BD Biosciences, San Jose, CA) and Summit 5.2 software (BD Biosciences, San Jose, CA) at the cell and immunology core in University of Missouri, Columbia.
Cyan adp flow cytometer
Manufactured by BD
Sourced in United States
The CyAn ADP flow cytometer is a high-performance instrument designed for multiparameter analysis of cells and other particles. It is capable of detecting and measuring various physical and fluorescent properties of samples, providing quantitative data on characteristics such as size, granularity, and the presence of specific molecules or markers.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm kit, by BD (3 mentions)
Influx cell sorter, by BD (2 mentions)
Donkey serum, by Bio-Rad (2 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Dead cell apoptosis kit with annexin 5 alexa fluor 488 propidium iodide, by Thermo Fisher Scientific (1 mentions)
Fc block, by BD (1 mentions)
Facsaria 2 sorp cell sorter, by BD (1 mentions)
Ambion ribopure rna purification kit, by Thermo Fisher Scientific (1 mentions)
Facsaria 2 cytometer, by BD (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Amd3100, by Merck Group (1 mentions)
Fty720, by Cayman Chemical (1 mentions)
Th1 th2 th17 cytometric bead array kit, by BD (1 mentions)
Fcap array software, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Fcs express software, by De Novo Software (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Facs buffer, by BD (1 mentions)
Anti gr 1 antibody clone rb6 8c5, by BioLegend (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (1 mentions)
18 protocols using cyan adp flow cytometer
1
Flow Cytometry Protocol for Cell Staining
2
Neuroblastoma Cell Cycle Analysis
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Neuroblastoma cells were fixed on ice with 70% ethanol, then washed twice in phosphate-citrate buffer (0.2 m disodium phosphate and 0.1 m citric acid), and resuspended in PBS containing 2 μg/ml propidium iodide, 0.1% Nonidet P-40, and RNase followed by analysis on BD CyAn ADP flow cytometer.
3
Flow Cytometric Characterization of Cells
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Cells were dissociated into single cells using TrypLE for 3 min at 37 °C, washed with 0.1% BSA-PBS, and either fixed with 1% paraformaldehyde and kept at 4 °C for maximum 1 week or immediately stained for flow cytometry. For the staining, after a wash with PBS, cells were blocked with 10% donkey serum (Bio-Rad) for 30 min at room temperature. Cells were then stained with the relevant conjugated antibodies diluted in PBS for 1 h at room temperature, protected from light (Table 1 ). Following two washes with PBS, cells were analysed using the Cyan ADP flow cytometer or sorted on the BD Influx cell sorter. Data was analysed using the FlowJo VX software.
Antibodies used for flow cytometry
| Target | Supplier | Clone |
|---|---|---|
| CD34 | Biolegend | 581 |
| CD43 | BD Biosciences | 1G10 |
| CDH5 | Biolegend | BV9 |
| CD44 | Biolegend | BJ18 |
| CD90 | Biolegend | 5E10 |
| CD31 | Biolegend | WM59 |
4
Quantification of TLR4 Expression in BIA-ALCL
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Expression of TLR4 in the three BIA-ALCL cell lines (TLBR-1, -2, -3) was examined by flow cytometry using anti-human CD284 (TLR4; BioLegend, San Diego, CA, USA). The human TLR4 stable cell line (Novus Biologicals; NBP2-26268, Littleton, CO, USA), expressing full-length human TLR4 with an N-terminal hemagglutinin (HA) tag, was used as a positive control. The TLR4 cell line was grown in DMEM with 10% FBS ± Blasticidin and all cell lines were grown with or without LPS. TLBR and control cell lines were stained with a Zombie NIR fixable viability dye and PE-conjugated anti-TLR4 and then fixed. Fixed cells were permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences) and stained intracellularly with anti-TLR4. Flow cytometry was performed on a Becton Dickinson CyAn ADP flow cytometer (BD Biosciences).
5
Isolation and Characterization of Thymic T Cells
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T lymphocytes were mechanically dissociated from thymus using frosted glass slides in DMEM with 5% FBS. Red blood cells were lysed using ACK buffer, and passed through 40 μm mesh filter. T cells were incubated with Fc Block (BD Biosciences) in 3% fetal bovine serum (FBS) (v/v) in PBS for 20 minutes on ice. 1–2 million cells were incubated with or without fluorescent-conjugated antibodies that recognize CD4, CD8, CD45, B220, or isotype control antibodies (BD Biosciences) in the dark for 30 minutes at 4°C. Cells were washed 3x and resuspended in 1% FBS (v/v) in PBS. For FACS sorting, CD45 stained cells were sorted using BD FACSAria II SORP cell sorter (BD Biosciences). CD45+ T cells and CD45- thymic stromal cells were subjected to RNA extraction using Ambion RiboPure RNA purification Kit (Thermo Fisher Scientific) and RT-PCR (Supplementary Methods). For CD4 and/or CD8-stained cells, they were fixed with paraformaldehyde at a final concentration of 1% (v/v). Cells were then run on Dako CyAn ADP flow cytometer or BD FACSCanto II Analyzer, and analyzed using FlowJo software (FlowJo, LLC., Ashland, OR). At least 30,000 viable events were collected for analysis.
6
Cell Cycle Analysis by Flow Cytometry
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To determine the percent of cells in each phase of the cell cycle (G1, S or G2/M), EV and ATIP1 stably transfected LNT-229 and LN-308 cells were seeded in 60 mm plates. After 48 h the cells were washed in PBS, trypsinized and washed again with PBS. The cells were fixed in 70% cold ethanol for 16 h, washed twice with PBS and flow cytometry buffer and incubated in dark for 30 min with propidium iodide (PI)/RNase A buffer (50 µg/mL PI, 100 µg/mL RNase A in flow cytometry buffer. The cells in different phases were analyzed using a CyAn ADP flow cytometer and FlowJo software (FlowJo LCC, BD Life Sciences, Heidelberg, Germany).
7
Mobilization of Mesenchymal Stem Cells
8
Cytokine Profiling of Cell Culture Supernatants
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After 6 days of culture, prior to the addition of radioisotope, cell culture supernatants were removed and frozen at − 80 °C. Cytokines were measured in the thawed culture supernatants using a Th1/Th2/Th17 cytometric bead array (CBA) kit (BD Biosciences) according to the manufacturer’s instructions. Samples were analysed on a CyAn ADP flow-cytometer, and data analysis was performed using BD FCAPArray software. To determine agonist-specific cytokine induction, background levels from uRBC alone were subtracted. Selected plasma samples were also analysed with Th1/Th2/Th17 CBA kits according to the manufacturer’s instructions.
9
Apoptosis Detection via Flow Cytometry
10
Single-Cell Dissociation and Flow Cytometry
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Cells were dissociated into single cells using TrypLE for 3 minutes at 37 °C, washed with 0.1% BSA-PBS and either fixed with 1% paraformaldehyde and kept at 4 °C for maximum 1 week or immediately stained for flow cytometry. For the staining, after a wash with PBS, cells were blocked with 10% donkey serum (Bio-Rad) for 30 minutes at room temperature. Cells were then stained with the relevant conjugated antibodies diluted in PBS for 1 hour at room temperature, protected from light. Following two washes with PBS, cells were analysed using the Cyan ADP flow cytometer, or sorted on the BD Influx cell sorter. Data was analysed using the FlowJo VX software.
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