Annexin 5 fitc

Cell apoptosis assay was performed to investigate the effects of PTCSC3 overexpression and silencing on osteoblast and osteoclast apoptosis. Briefly, cells were harvested, prepared as single-cell suspensions in serum-free media, and adjusted to 3 × 10⁴ cells per ml. Cell suspensions were transferred to a 6-well plate with 2 ml cell suspension in each well and cultured for 36 h. After that, cells were digested with 0.25% trypsin, stained with Annexin V-FITC and propidium iodide (Dojindo, Japan), and subjected to flow cytometry to detect apoptotic cells.

Flow cytometry was used to detect TNF-α-induced BMEC apoptosis. According to the manufacturer’s instructions, BMECs were incubated in 100 μl of incubation buffer with 5 μl of Annexin-V-FITC (Dojindo, Japan) and 5 μl of propidium iodide (PI) protected from light at room temperature for 15 min. Then, the 400 μl of additional incubation buffer was added to the samples and analyzed by flow cytometry analysis (FACS, Beckton Dickinson, USA).

Macrophages were incubated with fluorescent‐tagged antibodies for flow cytometry as follows: anti‐Human CD163‐PE, anti‐Human IL10‐APC, and anti‐Human TGFβ‐BV421 (all Biolegend, USA). Then, macrophages were detected by flow cytometry (Beckman). ECC‐1 and Ishikawa cells (300,000 cells/well) were seeded in 6‐well plates and allowed to adhere to culture plates overnight. Cells were then treated with MPA, CM, and IL10/TGFβ for 48 h. The cells were then trypsinized with EDTA‐free trypsin and washed twice with cold PBS. The cells were re‐suspended in 100 μl of binding buffer and stained with 1 μl of annexin V‐FITC and 1 μl of PI working solution for 15 min at room temperature in the dark (Dojindo, Japan). Finally, the cell apoptotic level was determined using a flow cytometer (Beckman). As negative controls, isotype‐matched antibodies with corresponding fluorescent labels were used.

Cellular ROS determination was performed based on previously described [29 (link)]. Apoptosis was measured using the Apoptosis Detection Kit (Dojindo Laboratories) as the company recommended. Briefly, Kyse 450 (150) cells were treated with DpdtbA for 24 h. Following this, cells were collected, washed, and stained with annexin V-FITC and propidium iodide (PI) following the manufacturer's instruction (Dojindo Laboratories, Japan). The intracellular ROS assay was similar to the abovementioned protocol, except that H₂DCF-DA was used to stain the cells.

The HEK239T cells were transfected with the NF1 WT or NF1 mutant constructed plasmids at 70–80% confluency. After being transfected for 24 h, the cells were harvested and stained with Annexin V-FITC for apoptosis analysis via flow cytometry according to the manual of an Annexin V-FITC Apoptosis Detection Kit (Dojindo, Japan).
After being transfected for 24 h, the cells were collected for immunoblotting. The cell lysates were collected according to our previously published protocol [7 (link)]. The protein was separated by 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA). The membrane was incubated with anti-Ras or anti-β-actin at 4°C overnight and incubated with anti-rabbit antibodies at room temperature for 1 h. The results of immunoblotting were visualized by chemiluminescence.