Cells were harvested at logarithmic period and washed twice with phosphate-buffered saline buffer (PBS). Each sample was re-suspended by 400 μL Binding Buffer (Biomiga, San Diego, CA, USA) at a density of 1.0×106 per well and stained with 5 μL AnnexinV-fluorescein isothiocyanate (AnnexinV-FITC) for 15 min at 4°C. Subsequently, 10 μL propidium iodide (PI) (BD Pharmingen, San Diego, CA, USA) was added. After transfected for 1 h, the positive cells were measured by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) with the dual excitation wavelength of 488 nm and 510 nm.
Binding buffer
Manufactured by Biomiga
Sourced in United States, China
Binding buffer is a solution used in various biomedical and molecular biology applications to facilitate the binding of target molecules, such as proteins or nucleic acids, to a solid support or matrix. It provides the appropriate pH, ionic strength, and other conditions necessary for efficient binding.
Automatically generated - may contain errors
3 protocols using binding buffer
1
Annexin V-FITC and PI Apoptosis Assay
2
Annexin V-FITC Apoptosis Assay
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For the apoptosis assays, the Annexin V-FITC Apoptosis Kit (ab14033) was purchased from Gemini Technology (Pudong, Shanghai, China) and the procedures using standard experiment methodologies were as follows: cells were transfected as described above and were harvested and resuspended in binding buffer (Biomiga, Haidian, Beijing, China). 80 μL of collected suspensions was added to FACS tubes and stained by the use of Annexin V-FITC and PI. Finally, the apoptosis rates of SKOV3 and CAOV3 cells were examined by applying a flow cytometer (Biosciences, Hangzhou, Zhejiang, China).
3
Apoptosis Analysis of BMMSCs and OMSCs
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Apoptotic BMMSCs and OMSCs in the α-MEM culture medium (Gibco; Thermo Fisher Scientific, Inc.) and adherent cells were collected and washed with PBS. Cells were adjusted to a density of 1×106/ml and 1 ml cell suspension was centrifuged at 1,500 × g for 5 min at room temperature. Subsequently, the cells were resuspended in 500 µl binding buffer (Biomiga, Inc.). Apoptotic cells were stained with 5 µl Annexin V-FITC and 10 µl propidium iodide for 10–15 min at room temperature, and analyzed by FACSCanto II (BD Biosciences). Cells from the lower left, the lower right, the upper right and the upper left represent normal, early, late and dead cells, respectively. The apoptotic rate was calculated as the sum of early and late apoptosis.
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