AnnexinV/PI apoptosis-detection kits (BD Biosciences, Franklin Lakes, NJ) were adopted to detect apoptosis rate. Briefly, the cells were resuspended in binding buffer and incubated with 5 μL of fluorescein isothiocyante (FITC)–Annexin V and 5 μL of PI for 15 min, and the FACScan flow-cell flow system (Becton Dickinson, San Diego, CA, USA) was used for apoptosis detection.
Facscan flow cell flow system
Manufactured by BD
Sourced in United States
The FACScan flow cell flow system is a laboratory instrument used for cell analysis and sorting. It is designed to detect and measure various characteristics of cells or particles suspended in a fluid stream, such as size, granularity, and fluorescence. The core function of the FACScan is to provide researchers with a tool for analyzing and sorting cell populations based on their specific properties.
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5 protocols using facscan flow cell flow system
1
Apoptosis Detection by Annexin V/PI
2
Annexin V-APC Apoptosis Assay in MCF-7/ADR Cells
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Annexin V-allophycocyanin (APC) apoptosis detection kit (BD Pharmingen, San Jose, USA) was used to detect apoptosis in transfected MCF-7/ADR cells. The cells were washed twice with precooled PBS buffer with a 1 × Binding Buffer used to prepare a 1 × 106 cells/mL suspension. 100 μL of cells was added to the flow tube with an appropriate amount of Annexin V and nucleic acid dye, gently mixed well, and placed in a dark place at room temperature for 15 minutes. Cells were washed once using 1 × Binding Buffer, and the supernatant was removed. 100 μL of 1 × Binding Buffer was used to dissolve 0.5 μg of SAv-FITC reagent before adding to the flow tube and gently mix well. 5 μL of PI was added and left at room temperature in the dark for 15 min. 400 μL of 1 × Binding Buffer was added to each assay tube, and the results were measured by a FACScan flow cell flow system (Becton Dickinson, San Diego, CA, USA) within 1 h.
3
Annexin V-APC Apoptosis Assay for A549 Cells
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The Annexin V-allophycocyanin (APC) Apoptosis Detection Kit (BD Pharmingen, San Jose, USA) was used to detect A549 cell apoptosis. A549 cells were washed twice with precooled PBS buffer and then suspended by 1 × Binding Buffer to 1 × 106 cells/ml. The EP tube was added 100 μl of cell suspension and Annexin V and nucleic acid dye to lightly mix till even. After placing it in a dark place at room temperature (20~25°C) for 15 min, we added 1 × Binding Buffer to wash the cells once, then removed the supernatant, and took 100 μl of 1 × Binding Buffer to dissolve 0.5 μg of SAv-FITC reagent, added it to the cell tube to mix gently. Hereafter, 5 μl PI was added and placed in a dark place at room temperature (20-25°C) for 15 min. After adding 400 μl of 1 × Binding buffer to each group of tubes, the FACScan flow cell flow system (Becton Dickinson, San Diego, CA, USA) was performed within 1 h to determine cell apoptosis.
4
Apoptosis Detection by AnnexinV-APC
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By using an AnnexinV-allophycocyanin (BD Pharmingen, SanJose, USA) apoptosis detection kit, apoptosis was detected. Cells were trypsinized and centrifuged, and the cells were washed twice using pre-cooled sterile PBS buffer. 1 × Binding Buffer was used to prepare cells of 1 × 106 cells/ml. Operate in strict accordance with the instructions for use of the kit, add AnnexinV and nucleic acid dye into the cells, gently mix them, and place them in darkness at room temperature for 15 minutes; add 5 µl PI, and place them in darkness at room temperature for 15 minutes. They were subsequently tested on a FACScanflow cell flow system (BectonDickinson, SanDiego, CA, USA).
5
Quantitative Chondrocyte Apoptosis Assay
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Annexin V-allophycocyanin apoptosis detection kit (556547, BD pharmacingen, San Jose) was adopted to detect chondrocyte apoptosis. Falcon tubes were taken, and NC tubes and tube numbers of specimens were arranged according to the sequence of specimens. Chondrocyte suspension (1 × 106 cells/mL) was prepared with 1×Binding buffer and 100 μL of which was added into Falcon tube. Next, annexin V and nucleic acid dye were also added, mixed gently, and reacted in the dark at ambient temperature (20-25°C; 15 min). Annexin V-biotin reagent was used. Cells were washed with 1×binding buffer once to remove the supernatant. Then, Sav-FITC (0.5 μg) was dissolved in 1×binding buffer (100 μL), which was added into the cell tube and mixed gently. Next, cells were dyed with 5 μL propidium iodide in the dark at ambient temperature for 15 min. Each test tube was added with 400 μL of 1×binding buffer, and apoptotic cells were calculated by means of FACScan flow cell flow system (Becton Dickinson, San Diego, CA, USA) within 1 h.
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