Accuri c6 flow cytometry

Cell-cycle distribution by PI staining was performed as previously described17 (link). Briefly, 50,000 cells were cultured in 6-well plates, treated for 72 h with concentrations ranging from 1 to 50 μM, collected by centrifugation, fixed in cold 70% ethanol and stained with a PBS solution containing PI (62.5 mg/ml; Sigma-Aldrich) and RNase A (1.125 mg/ml; Sigma-Aldrich). Samples were acquired with C6 Accuri Flow cytometry (BD Bioscience) and the percentage of cells in the different phases of cell cycle and in the sub-G1 compartment was calculated. Apoptosis was quantified by cytofluorimetric analysis staining cells simultaneously with FITC-annexin V and the nonvital dye PI (Immunological Sciences, Rome, Italy), following the manufacturer’s instruction. At the end of incubation with the respective reagents, samples were analyzed with C6 Accuri Flow cytometry (BD Bioscience). About 20,000 events were acquired and gated using forward scatter and side scatter to exclude cell debris. Bivariate analysis allows the discrimination of viable cells (FITC−/PI−), early apoptotic (FITC+), and late apoptotic or necrotic cells (FITC+/PI+).

Apoptotic cells were stained with Annexin V FITC detection kit (BD Bioscience) according to the manufacturer's protocol. Briefly, cells were trypsinized and washed with ice‐cold phosphate‐buffered saline (PBS) before suspended in Annexin V binding buffer. Staining of 10⁵ cells was performed with 5 μL Annexin V solution and 5 μL propidium iodide (PI) solution at room temperature for 15 minutes, and samples then were subsequently applied to flow analysis by Accuri C6 Flow Cytometry (BD bioscience) for collection the emission data from FL1 channel and FL2 channel. For cell cycle analysis, 2 × 10⁶ cells were collected and fixed in 70% ethanol at 4°C overnight. Cells were washed twice with PBS and stained with 500 μL of PI staining solution (50 μg/mL PI and 100 μg/mL RNase in PBS) for 30 minutes in dark. Stained cells were then performed cell cycle analysis using Accuri C6 Flow Cytometry by collecting emission data from FL2 channel at 575 nm.

EC apoptosis was assayed using the FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, Cat # 556570) following the manufacturer-recommended protocol. Briefly, cells were collected using 0.05% Trypsin-EDTA, washed once with PBS and resuspended in the binding buffer. Cells were then stained with 5 μL Propodium Iodide Staining solution and 5 μL FITC Annexin V and incubated in the dark at room temperature of 20 min. Following incubation, cells were immediately analyzed on Accuri C6 flow cytometry (BD Biosciences).

Cells (30 × 104 cells/well) were seeded in 6-well plates. After exposure to different treatment, cells incubated in propidium iodide/Triton X-100 staining solution with RNase A for 10 min at room temperature. Samples were analyzed by Accuri C6 Flow Cytometry (BD Biosciences, Franklin Lakes, NJ, USA). And its cell cycle-dependent distribution was analyzed using the ModFit LT 3.0 software.

NRK cells were seeded in an eight-linked confocal dish at an appropriate density (about 6 × 10³/cm²) and cultured with AuNPs for 24 h. Discard the culture medium, and then stained with LysoSensor™ Green DND-189 (1:1000) (Thermo, L7535) at 37 °C for 30 min. After washing, cells were observed with LSM710 confocal microscopy (Zeiss) with ×63/1.40 NA oil objective. The excitation wavelength was FITC 488 nm. At the same time, flow cytometry was used to semi-quantitatively analyze the above fluorescence intensity. LysoSensor™ Green DND-189 stained cells were collected. The intracellular fluorescence intensity was detected by Accuri C6 Flow Cytometry (BD Biosciences). Detect channel: FL1, 488 nm, 533 nm/30 nm.

MMP (ΔΨm) levels were measured using rhodamine 123 (Sigma-Aldrich Co.; Ex/Em = 485 nm/535 nm) and JC-1 dyes (Enzo Life Sciences ; Ex/Em = 515 nm/529 nm). Briefly, 5 × 10⁴ cells in 12 well culture plate (BD Falcon) were incubated with the indicated concentration of SAHA for 24 hours. Cells were washed twice with PBS and incubated with 0.1 μg/ml rhodamine 123 or 10 μg/ml JC-1 at 37^°C for 30 min. For staining nucleus, cells were incubated with 500 nM 4′, 6′-diamidino-2-phenylindole (DAPI, Life Technologies, Ex/Em = 358 nm/461 nm) at 37^°C for 30 min. The intensity of rhodamine 123 staining was determined by Accuri C6 flow cytometry (BD Sciences). The negative staining of rhodamine 123 from cells indicated the loss of MMP (ΔΨm) in cells. After incubation with JC-1 and DAPI, cells were washed three times with PBS and images were collected by using a fluorescence microscope (FLoid^® Cell Imaging Station, Life Technologies) in × 400 magnification. Green fluorescence indicates a monomer at low ΔΨm and red fluorescence presents high ΔΨm.