Accuri c6 plus

Human pancreatic cancer cells (MIA PaCa-2 cells, Korean Cell Line Bank, Seoul, Korea) were maintained in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. To visualize the intracellular delivery efficiency of VP-DOX-PLs, MIA PaCa-2 cells (2 × 10⁵ cells per well) were seeded in confocal dishes. Doxorubicin and verteporfin were treated at the concentration of (200 and 100) ng/mL, respectively, and incubated for 24 h. The cells were washed twice using cold PBS and fixed using 4% paraformaldehyde for 15 min. Then, the cells were stained with DAPI solutions, and fluorescence images of DOX-PLs, VP-PLs, and VP-DOX-PLs were obtained using confocal laser scanning microscopy (LSM 880, Zeiss, Jena, Germany). For the quantitative analysis, the same experiment was conducted with Accuri C6 Plus (BD Accuri C6 Plus, BD, Piscataway, NJ, USA), and 10,000 cells were counted for each group. The fluorescence of doxorubicin was measured through the FITC filter, and the fluorescence of verteporfin was measured using an APC filter.

An Annexin V-FITC Apoptosis Detection Kit (AD10; Dojindo Laboratories, Kumamoto, Japan) was used to analyze MDA-MB-231 cell apoptosis. Briefly, the cells were seeded into Costar^® 6-well plates (Corning Inc.) and digested with 0.05% trypsin after treatment with the drugs for 24 h, then collected and washed twice with ice-cold phosphate-buffered saline (PBS, 1×; Hyclone Laboratories Inc., Logan, UT, USA). Thereafter, a 1× Annexin V binding solution was added to make a cell suspension at a final concentration of 1 × 10⁶ cells/ml. The cells were then stained with Annexin V-FITC and propidium iodide (PI) for 15 min in the dark at room temperature (RT). After adding 400 µl of 1× Annexin V binding solution into each tube of the cell suspension, the cells were loaded onto a flow cytometer (Accuri C6 Plus; BD BioSciences, Franklin Lakes, NJ, USA) within 1 h. The results of three independent experiments were analyzed using BD Accuri C6 Plus software, according to the manufacturer’s instructions.

To detect the apoptosis of C-33A and HeLa cells, flow cytometry experiments were conducted using apoptosis detection kit (Dojindo Laboratories, Japan). After treatment with drugs for 24 h, C-33A and HeLa cells were digested with 0.05% trypsin (Gibco, ThermoFisher Scientific), and then collected and washed with ice cold PBS two times; 1× Annexin V Binding Solution was added to make a cell suspension with a concentration of 1 × 10⁶ cells/ml. The cells were then stained with Annexin V, fluorescein isothiocyanate (FITC), and propidium iodide (PI) for 15 min at room temperature (RT) before 400 µl 1× Annexin V Binding Solution was added. The cells were then loaded to a flow cytometer (Accuri C6 Plus, BD BioSciences, United States) within 1 h. Results were analyzed using BD Accuri C6 Plus software.

Isolated B cells were isolated from HIV+/- subjects. Portion of isolated B cells was incubated with CD19 antibody (1/200 of stock, Milteny) for 30 mins to determine the purity using BD Accuri C6 Plus with corresponding optical filters. The rest of B cells was washed and fixed with 4% paraformaldehyde at RT for 20 mins. Pelleted cells were washed and permeabilized with saponin-containing 1 × Perm/Wash buffer (BD Biosciences) as described [15 (link)]. Cells were incubated with an anti-PLK1 antibody (200μg/ml) diluted in 1 × Perm/Wash buffer for overnight at 4°C, followed by incubation with the fluorophore-conjugated secondary antibody for 1 hr at RT in the dark. The staining buffer (1 × D-PBS with 2% bovine serum albumin [BSA]) was added to resuspend the cells, followed by flow cytometry analysis using the BD Accuri C6 Plus with corresponding optical filters. For the Jurkat and BJAB co-culture assays, BJAB cells were labeled separately with CFSE (Thermo Fisher) for 30 mins according to instruction and incubated with HIV-1 IIIB infected Jurkat cells. These cells were incubated with anti-PLK1 antibody, and subsequently the fluorophore-conjugated secondary antibody. The mean fluorescence intensity (MFI) and the percentage of fluorescence-positive cells were determined by using the FlowJo V10 software.

Briefly, MDA-MB-231 cells (1 × 10⁶) were seeded in 6-well plates and after 24 h cells were treated with DMSO (3 µL/mL), paclitaxel (20 nM and 200 nM), XAV939 (10 µM), and paclitaxel + XAV939 (20 nM + 10 µM) for 48 h. After trypsinization, cells were fixed with 70% ethanol and incubated at 4 °C for 30 min. Next, cells were washed and suspended in PBS containing 0.5 mg/mL ribonuclease A (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 30 min at 37 °C. Cells were stained with 50 μg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) and analyzed with BD Accuri C6 Plus (BD) at an excitation wavelength of 488 nm and an emission wavelength of 630 nm. The cell cycle distribution was quantified by using BD Accuri C6 Plus software.

Reactive oxygen species level of HSCs, HPCs and BMNCs was tested by the flow cytometry with 2,7‐dichlordihydrofluorescein diacetate (H2DCFDA, Invitrogen), as the ROS probe after IR exposure. BM lineage negative haematopoietic cells (Lin–cells) were stained with anti‐Sca‐1‐PE and anti‐c‐kit–Alexa Fluor 700 antibodies and then added with 5 μM H2DCFDA and incubated for 30 min at 4°C. Cells were washed twice with PBS and resuspended in FACS buffer (PBS +0.1% FBS). Mean fluorescence intensity (MFI) was measured on a flow cytometer (BD Accuri™ C6 Plus).
The HSCs protein expression level of NOX4, γ‐H2AX, p‐P38 and Nrf2 was evaluated by the flow cytometry. Bone marrow cells were first stained with a c‐kit antibody and fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences, USA) before being stained with the primary antibody including anti‐NOX4²² (14347‐1‐AP, Proteintech), γ‐H2AX²³ (Cell Signaling Technology), anti‐p‐P38 antibody²⁴ (BD Pharmingen) (1:50), anti‐Nrf2²³ (ab62352, Abcam) at room temperature. The diluted secondary antibodies were added and incubated at room temperature. Then, the cells were resuspended in FACS buffer and measured on a flow cytometer (BD Accuri™ C6 Plus).