The apoptosis incidence was determined by an Amnis Flow Sight flow cytometer (Merck & Co., Inc.) using the Annexin V-FITC Apoptosis Detection kit (cat. no. 556547; BD Biosciences). Briefly, B16 cells were incubated with fresh medium (as control) or medium containing PLP for 48 h at 37°C. After treatment, the cells were collected by centrifugation at 303 × g for 5 min at 24°C, washed twice with ice-cold PBS, suspended in 1X Binding Buffer and incubated with 5 µl each of propidium iodide (PI) and Annexin V-FITC solutions. Probes were incubated in darkness at 37°C for 15 min. Samples were examined with a flow cytometer and quantified using Amnis IDEAS software v6.1 (Merck & Co., Inc.). The number of total apoptotic cells, including early and late apoptotic cells, was counted and represented as percentages of the total cell count.
Amnis flowsight flow cytometer
Manufactured by Merck Group
Sourced in United States
The Amnis FlowSight flow cytometer is a laboratory instrument designed for cell analysis. It utilizes flow cytometry technology to rapidly measure and analyze the physical and chemical characteristics of cells or other particles suspended in a fluid stream.
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4 protocols using amnis flowsight flow cytometer
1
Quantifying Apoptosis in B16 Cells
2
EGFR and c-Src Antibody Labeling
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Cells were incubated with a PE-conjugated anti-EGFR antibody or an unlabeled anti-cSrc antibody for 1 h in PBS containing 1% FBS. Cells were rinsed with the same buffer and further stained with a FITC-conjugated secondary anti-cSrc antibody for 1 h. Isotype-matched, conjugated antibodies were used for control staining. Cells were analyzed using an Amnis FlowSight flow cytometer (EMD Millipore, Billerica, MA, USA). Data analysis was performed using the FlowJo program.
For cell sorting, cells were dissociated using Accutase (STEMCELL Technologies) for 5 min and cells rinsed with using 3% BSA in PBS. Then, 1 µg of EGFR-PE antibody was added to a suspension of 1 × 106 cells and incubated for 1 h at 4 °C. Next, the unbound antibody was washed away three times using the same buffer. Cells were sorted with a FACSAria II instrument (BD Bioscience) and used for subsequent experiments.
For cell sorting, cells were dissociated using Accutase (STEMCELL Technologies) for 5 min and cells rinsed with using 3% BSA in PBS. Then, 1 µg of EGFR-PE antibody was added to a suspension of 1 × 106 cells and incubated for 1 h at 4 °C. Next, the unbound antibody was washed away three times using the same buffer. Cells were sorted with a FACSAria II instrument (BD Bioscience) and used for subsequent experiments.
3
HeLa Cells Transfection and GFP Analysis
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HeLa cells transfected with pCXB-EmGFP plasmid were analysed for green fluorescent protein expression by flow cytometry. 2 days after transfection, cells were detached from cell culture vessels to a single-cell suspension with TrypLE Select (Life Technologies) and analysed using Amnis FlowSight flow cytometer (EMD Millipore). The acquisition was set for 70,000 events per sample, and the data were analysed using IDEAS software (EMD Millipore).
4
Flow Cytometric Analysis of Macrophage Phenotypes
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RAW264.7 macrophages were collected after mechanical stretch, washed, and centrifuged with cold buffer solution. The cells were resuspended with 5 μl PE-CD86, 5 μl FITC-CD11b, and 100 μl combined buffer, respectively, and incubated for 30 min shielded from light. Then, the cells were fixed and permeabilized using Fixation & Permeabilization Kit (Fcmacs, Nanjing) according to the instructions. Subsequently, APC Rat anti-CD206 was added. After 15 min of incubation, the cells were washed and then analyzed by Amnis FlowSight flow cytometer (Merck Millipore, USA).
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