Annexin 5 fitc pi apoptosis detection kit

After the MH-S cells were treated according to experimental groupings, 1×10⁶ cells were collected by centrifugation, resuspended in PBS, and assayed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). Changes in intracellular Ca²⁺ concentration were detected using Fluo-4 AM Cell Membrane Permeable Calcium Ion Fluorescent Probe (#40704ES50, YEASEN, Shanghai, China). Cells were co-incubated with Fluo-4 AM for 30 min in a 37 °C incubator, and washed with HBSS solution 3 times. HBSS solution was added to cover the cells, incubated for 30 min in a 37 °C incubator, and then flow cytometry detection was performed. Apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit (#40305ES20, YEASEN, Shanghai, China). Cells were incubated with Annexin V-FITC and PI Staining Solution at room temperature for 15 min in the dark, then mixed with Binding Buffer and placed on ice, followed by the assay on the flow cytometer. Macrophage polarization was detected by CD86 and CD206 antibodies that labeled M1 or M2 macrophages, respectively. Cells were co-incubated with anti-CD86 and anti-CD206 antibodies at room temperature for 30 min and washed with PBS, then incubated with Alexa Fluor^® 488/594-coupled secondary antibodies for 30 min at room temperature and then assayed on the flow cytometer.

CPT was obtained from Meryer Co., Ltd., 3,3'‐Thiodipropionic acid, 1‐ethyl‐3(3‐dimethylpropylamine) carbodiimide (EDCI) and 4‐Dimethylaminopyridine (DMAP) were purchased from Macklin Co., Ltd., Methoxypolyethylene glycol (PEG2000) and Hoechst 33 342 were obtained from Aladdin Biochemical Technology Co., Ltd. DSPE‐PEG2000 was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. AZD4635 was obtained from MedChemExpress (MCE). Cy5.5 (Cyanine5.5)‐carboxylic acid was purchased from Nanjing Goyoo Biotech Co.,Ltd. 96‐well, 12‐well, 6‐well plate were obtained from NEST Biotechnology. MTT and FITC‐phalloidine were purchased from Solarbio life sciences. Annexin V‐FITC/PI Apoptosis Detection Kit and d‐Luciferin sodium salt were obtained from Yeasen Biotech Co., Ltd. Mouse Adenosine and ATP ELISA Kit were purchased from Shanghai Jianglai Biotechnology Co., Ltd. The antibodies of CRT, HMGB1 and Caspase 3 were purchased from Abcam. The antibody of GAPDH and HRP labeled sheep anti‐rabbit secondary antibody were obtained from BIOSS.
The synthesis and characterization of TST and CPT‐S‐PEG can be found in the Supporting Information.

Apoptotic cells labeled with Annexin V-FITC/PI were used for the evaluation of phagocytosis by flow cytometry, as described in Annexin V-FITC/PI Apoptosis Detection Kit (Cat No. 40302, YEASEN, Shanghai, China). Briefly, the pretreated cells were collected and centrifuged at 300g at 4°C for 5min, then precooled and washed twice with PBS. Cells from 1 to 5×10⁵ were collected. PBS was discarded and 100 μl of 1×Binding Buffer was added to resuspend the cells. Add 5 μl Annexin V-FITC and 10 μl PI staining solution and mix gently. After adding 1×Binding buffer, the samples were darkened and kept at room temperature for 10-15 min. The samples were mixed and detected by flow cytometry within 1h. The cells were analyzed and sorted using a BD FACS Aria™ II Cell Sorter (BD Biosciences, Franklin Lakes, NJ, USA). The data obtained were analyzed using FlowJo version 10.6 software (FlowJo, LLC, Ashland, OR, USA).

Cell apoptosis was analyzed by an Annexin V-FITC/PI apoptosis detection kit (Yeasen Biotech, China). Cells were harvested and then washed twice with PBS. The cell pellet was suspended in 1× binding buffer and supplemented with Annexin V-FITC and PI. The cells were gently vortexed and incubated for 15 min at room temperature in the dark, then supplemented with 400 μL 1× binding buffer and submitted to flow cytometry analysis using a FACSCalibur flow cytometer equipped with Cell Quest software (BD Biosciences, USA).^{59 (link)}

Cell samples were prepared using an Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Cat:40302ES50). Briefly, six-well plate cells were digested with EDTA-free 0.1% trypsin. The cells were washed twice with ice-cold PBS and centrifuged for 5 min at 500 × g at 4°C. The supernatant was discarded and the cell pellets were resuspended in 1× binding buffer. The cell suspension (100 μL) was added to 5 μL of Annexin V-FITC solution and 10 μL of PI solution and mixed gently. After 15 min of reaction at room temperature in the dark, 400 μL of 1× binding buffer was added. The samples were analyzed by flow cytometry within 1 h.