The cells were seeded into 6-well-plates overnight. After serum-free starvation treatment for 48 h, the cells were collected, stained with Annexin V–APC/7-aminoactinomycin D (7-AAD) (MultiSciences, Hangzhou, China) following the manufacturer's instructions and detected with a BD FACSCalibur flow cytometer (Becton Dickinson, USA).
Annexin 5 apc 7 aminoactinomycin d 7 aad
Manufactured by MultiSciences Biotech
Sourced in China
Annexin V-APC/7-aminoactinomycin D (7-AAD) is a laboratory reagent used in flow cytometry analysis. Annexin V is a protein that binds to phosphatidylserine, a component of the cell membrane. 7-AAD is a DNA-binding dye. Together, these two reagents are used to detect and quantify apoptosis, or programmed cell death, in cell samples.
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3 protocols using annexin 5 apc 7 aminoactinomycin d 7 aad
1
Apoptosis Analysis by Flow Cytometry
2
Apoptosis and Cell Cycle Analysis
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The transfected cells were seeded onto a 6-well plate at a density of ~1 × 105 cells per well. After 24 h, the cells were harvested with trypsin and washed three times with chilled PBS buffer. The cells were then stained with Annexin V-APC/7-aminoactinomycin D (7-AAD; MultiSciences, China) at room temperature in the dark. All procedures followed the manufacturer’s protocols. Then, an FAC Scan flow cytometer (Becton–Dickinson, USA) was used to analyze the samples and measure the percentage of apoptotic cells.
Cell cycle analysis was also used to further evaluate cell proliferation. Briefly, cells were fixed with chilled 70% ethanol and stored at −20 °C overnight. The next day, the cells were centrifuged after the supernatant was discarded, rewarmed, and stained with PI (50 μg/mL; MultiSciences, China) for 30 min before flow cytometry. More than 3 × 104 cells were detected.
Cell cycle analysis was also used to further evaluate cell proliferation. Briefly, cells were fixed with chilled 70% ethanol and stored at −20 °C overnight. The next day, the cells were centrifuged after the supernatant was discarded, rewarmed, and stained with PI (50 μg/mL; MultiSciences, China) for 30 min before flow cytometry. More than 3 × 104 cells were detected.
3
Apoptosis Analysis of BSGLWE Treatment
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Apoptosis was calculated using the reagent Annexin V-APC/7-aminoactinomycin D (7-AAD; Multi-Sciences, Hangzhou, Zhejiang, China). Cells were seeded into a six-well plate at a density of approximately 2–3×105 cells per well and treated with different concentrations of BSGLWE for 24 hrs. Then, the cells were harvested with trypsin and pelleted by centrifugation. After being washed 3 times with PBS, the cells were incubated with Annexin V-APC/7-AAD kit for 15 min at normal temperature in the dark. All procedures followed the manufacturer’s protocols. Then, the samples were measured and analyzed by a flow cytometer (FACSCalibur, BD, San Jose, CA, USA). Annexin V-APC (+)/PI (−) and Annexin V- APC(+)/PI(+) staining signified cells that were in the early and later stages of apoptosis, respectively.
The Hoechst 33342 nuclear staining method was used to measure morphological changes in apoptotic cells. First, 4×105 cells per well were seeded into a six-well plate and treated with different concentrations of BSGLWE for 24 hrs, then cells were cultured with Hoechst 33342 for approximately 15 mins. At last, a fluorescence microscope (Olympus, Tokyo, Japan) was equipped to visualize morphological changes of apoptotic cells at 365 nm.
The Hoechst 33342 nuclear staining method was used to measure morphological changes in apoptotic cells. First, 4×105 cells per well were seeded into a six-well plate and treated with different concentrations of BSGLWE for 24 hrs, then cells were cultured with Hoechst 33342 for approximately 15 mins. At last, a fluorescence microscope (Olympus, Tokyo, Japan) was equipped to visualize morphological changes of apoptotic cells at 365 nm.
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