Fc500

Briefly, following the manufacturer’s instructions, using 2,7-dichlorodihydrofluorescein diacetate and dimethyl sulfoxide as stock solutions, intracellular ROS levels were measured as described previously (Zhou et al., 2017 (link)). Before treatment, SH-SY5Y cells were seeded in 6-well plates for at least 12 h. Incubation with or without reserpine (100 μmol/L) was for 24 h followed by 3 washes with cold PBS after replacing the medium with DCFH-DA (25 mol/L). The fluorescence intensity was assessed using a Zeiss fluorescence confocal microscope LSM 800 (Oberkochen, Germany). The ZEN software was used to analyze the images. To perform quantitative flow cytometry (FC500; Beckman Coulter, Brea, CA) analysis, adherent cells were treated with reagents and fluorescent probes, followed by suspension in 500 µl PBS. Measurement and analysis of fluorescence intensity were performed with Cxp (FC500; Beckman Coulter).

The transfection efficiency of the complexes was measured by flow cytometry (FC500, Beckman-Coulter, USA). A549 cells were seeded at a density of 1 × 10⁶ cell/well in 6-well plates. The transfected method was as described above. The cells were trypsinized, washed, and resuspended in PBS. The expression levels of GFP were determined by flow cytometry (FC500, Beckman-Coulter, USA) within 30 min.

hUCMSCs were cultured in 6-well plates and either treated with LPS (0, 1, 40 or 50 μg/ml) for 24 h, or pretreated with LPS (0, 0.1, 1, 10 or 20 μg/ml) for 12 h with subsequent exposure to 50 μg/ml LPS for 24 h. Following treatment, cells were harvested using trypsin-EDTA and collected by centrifugation at 900 × g for 5 min at room temperature. Cells were washed, resuspended in PBS, and labeled with Annexin V and propidium iodide (PI; from the BD Apoptosis Detection kit) for 20 min. Analyses were performed by flow cytometry (FC500; Beckman Coulter, Brea, CA, USA) using FC500 MPL CXP2.1 software.

Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll–Paque (Pharmacia) density-gradient centrifugation. After blocking with FcR, PBMCs were incubated with diluted antibodies for phenotyping and then analyzed using flow cytometry (FC-500, Beckman Coulter, FL, USA). Data analysis was conducted using FlowJo software (version 7.6.2, Tree Star, Ashland, OR, USA). Circulating T lymphocyte subgroup and NK cells were identified by multiparameter flow cytometry according to the previous description [18 (link), 19 (link)]. T lymphocyte subsets were identifying CD4-FITC/CD8-PE/CD3-PC5, and NK cell was identifying with CD3-FICT/CD(16+56)-PE (BD Biosciences, CA, USA). The phenotype of NK cells was CD3-CD16+CD56+. Four-color flow cytometry (FC-500, Beckman Coulter, FL, USA) was performed to determine the phenotypes of T regulatory cells (Tregs) using the CD3-PC5, CD4-PE, CD25-FITC (BD Biosciences, CA, USA), and CD127-PC7 antibodies (BioLegend, San Diego, CA, USA). Tregs were defined as CD3+CD4+CD25+CD127− cells.

Cells (3 × 10⁵ cells/mL) were cultivated in 6-well plates for 24 h. Both the control and Tf-PEG liposomes were added to the 6-well plates, followed by sonication using a 35-kHz US bath for 5 min. The sonicated plates were then incubated for 1 h at 37 °C in a humidified atmosphere with 5% CO₂. Following the incubation period, each well was washed with PBS buffer and harvested using a trypsin solution. Harvested samples were then analyzed by measuring calcein fluorescence intensity using a flow cytometer [FC 500 (Beckman Coulter FC 500, US)]. In each insonation experiment, the sonolysis studies (the effect of US on cell viability) were performed using the Trypan blue exclusion assay. A minimum of three independent assays was performed for each treatment.

Cell proliferation was measured by flow cytometry using the CellTrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific). Briefly, wild-type Jurkat cells were stained with 1 μM CFSE in PBS for 20 min, and unincorporated CFSE was quenched and washed with complete culture medium following the manufacturer’s protocol. Stained cells were incubated for 48 h before their transfection with the NS control siRNA and the siRNA against the ELOVL5. Cells were then analyzed by flow cytometry (FC500; Beckman-Coulter) at 48 and 96 h posttransfection.
Cell proliferation was also measured using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) in combination with the FxCycle Violet Stain (Thermo Fisher Scientific) following the manufacturer’s protocol. Briefly, at 72 and 96 h posttransfection, Jurkat cells were incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h at 37°C, washed, fixed, and permeabilized, and the Click-iT reaction was done to conjugate the incorporated EdU molecules to the Alexa Fluor 488. All components used were provided with the kit. Cells were then resuspended in 1 ml PBS and stained with the FxCycle Violet Stain following the manufacturer’s protocol before being analyzed by flow cytometry (FC500; Beckman-Coulter).