Lsrfortessa sorp cytometer

The Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Life Technologies) was used in combination with propidium iodide (PI, Sigma) staining for the calculation of proliferating cells and cell cycle phases using the flow cytometer in siRNA transfection experiments and in combined drug treatments (see Supplementary Extended Methods). A BD LSRFortessa SORP cytometer was used to measure the amount of Alexa Fluor 488 dye (EdU-positive cells) and PI (DNA content).

For flow cytometry analysis, differentiated THP-1 cells were pretreated with increasing concentrations of PEG35 and then co-incubated with PKH26-labeled ExoAP. Subsequently, cells were washed with PBS and trypsinized. Pelleted cells were fixated in 4% paraformaldehyde and incubated for 15 min at 4 °C. After incubation, cells were washed again with PBS and resuspended in 0.3 mL of PBS buffer for flow cytometry analysis. The PKH26 fluorescence was measured on a BD LSR Fortessa SORP cytometer using the FacsDiva software (BD Biosciences, San Jose, CA, USA). Data analyses were performed with FlowJo software (version 4.4.1; FlowJo LLC, Ashland, OR, USA).

Apoptosis was measured in siRNA transfection experiments and in combined drug treatments using the Annexin-V-Alexa Fluor 568 antibody (Roche and Life Technologies). This was combined with bis-benzimide dye (Sigma) staining for the detection of late apoptotic and necrotic cells (see Supplementary Extended Methods). A BD LSRFortessa SORP cytometer was used to measure the amount of Alexa Fluor 568 dye (Annexin-V-positive apoptotic cells) and bis-benzimide (late apoptotic/necrotic cells).

Cell suspensions were obtained from digested cardiac tissue samples of infected mice treated with MSCs, MSC_G-CSF, or saline, as previously described (16 (link)). The cell suspensions were allowed to pass through a 70 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) and counted with a hematocytometer. Aliquots of 10⁶ cells were used for each test tube and 1 µL of Fc blocking reagent (BD Biosciences) was added. Fluorochrome-conjugated antibodies used were: CD11b-PE-Cy5, CD45-APC and GR-1-FITC, or Ly6C-FITC and Ly6C-PE (BD Biosciences). Samples were incubated with the antibodies for 20 min at RT. Sample acquisition was performed using a BD LSRFortessa SORP cytometer using BD FacsDiva v.6.2. Acquired data were analyzed by FlowJo v7.5 (FlowJo Enterprise, Ashland, OR, USA). CD11b⁺GR-1⁺ MDSCs were sorted from digested hearts of MSC_G-CSF-treated mice or from the bone marrow, as indicated in Section “Results.” The cells were stained with GR-1-PE (BD Biosciences), CD11b-APC (ThermoFisher Scientific), and CD45-APC-Cy7 (BD Biosciences), using a FACS Aria cell sorter (BD Biosciences), achieving a purity of approximately 98%.

The concentration of intracellular Ca²⁺ concentration was measured using the fluorescence Ca²⁺ indicator Fluo-4 AM according to a previous report[19 (link)]. When cells began to grow exponentially, they were treated with 10 pulses of nsPEFs with 100 ns durations at 5 kV/cm, 10 kV/cm, 20 kV/cm electric fields. After nsPEFs treatment, the cells was washed with Hank's Balanced Salt Solution (HBSS) twice, and loaded with 4 μM Fluo-4 AM (Beyotime Institute of Biotechnology, Jiangsu, China) for 45 min in the dark. Then the cells were rinsed with HBSS twice and incubated for another 20 min at 37°C to ensure that Fluo-4 AM had completely transformed into Fluo-4 in the cells. Image were taken with a laser scanning confocal microscope. Detection of intracellular Ca²⁺ was carried out by a BD LSRFortessa SORP cytometer (BD Bioscience). The Ca²⁺ concentration was expressed as the mean fluorescence intensity of each treated group compared with that of the vehicle control group.