Fluo 4 am

The levels of free cytosolic Ca²⁺ were measured using the cell-permeable Ca²⁺-sensitive fluorescent dye Fluo-4 AM (Beyotime Biotechnology, Shanghai, China). Each experimental group of PMVECs was stretched for 4 h in Ca²⁺- and Mg²⁺-containing Hanks’ Balanced Salt Solution (HBSS; Solarbio Technology Co., Ltd., Beijing, China). PMVECs were pretreated with the extracellular calcium-ion chelating agent EGTA (2 mM; ApexBio Technology) for 1 h before 18% CS was performed. PMVECs were stretched for 4 h and then incubated with 5 µM Fluo-4 AM for 30 min at 37°C in the dark, and the cells were then washed. A vehicle control group was used to confirm equal loading of Fluo-4 AM. The control group was maintained in Ca²⁺- and Mg²⁺-containing HBSS without Fluo-4 AM. We measured Ca²⁺ levels after treatment with HC-067047 (300 nM), an antagonist of TRPV4, which was defined as minimum fluorescence intensity. The fluorescence intensities of the other groups are expressed as %min. The fluorescence intensity of the Fluo-4 AM probe was analyzed by using a CytoFLEX (Beckman Coulter, Inc.) flow cytometer, and the results were analyzed using CytExpert software.

The samples were separated into RBCs and WBCs. The RBC labeling antibodies were CD45-PE/Cy7 (IM3548, Beckman Coulter, USA), CD36-PE/Cy5 (Customized from BD Biosciences, USA), CD138-PE (A40316, Beckman Coulter, USA), and Fluo-4 AM (S1060, Beyotime, Shanghai, China), and the WBC labeling antibodies were CD45-PE/Cy 7, CD15-PE/Cy5 (B49217, Beckman Coulter, USA), CD11b-PE (IM2581U, Beckman Coulter, USA), and Fluo-4 AM. A Beckman Coulter FC500 flow cytometer was used to excite the fluorescence and collect the signal.