Accuri flow cytometry

After 72 h of PMMA exposure in the range of 0.25–1 mg/mL, the levels of TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-23, IL-33, MCP-1, INF-α, and IFN-γ were assessed by LEGENDPlex™ human inflammation panel (Biolegend, USA) using BD Accuri flow cytometry (BD Biosciences, USA) following the manufacturer’s protocol. The cell culture supernatant was used for the determination of cytokine secretion. The size of the beads is represented in the FL-3 channel, and the fluorescence intensity in the FL-2 channel correlates proportionally with cytokine concentrations (pg/mL). The cytokine concentration was calculated using the standard curve, and the results were normalized to the control group.

Apoptosis assay was performed using apoptosis assay kit (Invitrogen, USA). Briefly, HeLa, MCF-7 and RWPE-1 cells were seeded in 24 well tissue culture plates (about 2 × 10⁵ cells/well) and incubated for 24 h. Subsequently, culture medium was replaced with fresh medium containing 2.5 μM of LS10 along with a negative control (cells without peptide). Apoptosis induced by the peptide was examined by Annexin V/PI staining followed by flow cytometry analysis of stained cells. Briefly, after 2 h and 24 h of incubation with LS10, cells were harvested, washed twice with cold PBS and resuspended in 1X Annexin binding buffer. Cells were then transferred to fresh 1.5 ml tube containing 100 μl of binding buffer and 5 μl of FITC-conjugated Annexin V and 1 μl of PI were added. The cells were vortexed gently and incubated for 15 min at room temperature in dark. After incubation, 200 μl of 1X Annexin binding buffer was added to each tube and stained cells were analyzed by Accuri flow cytometry (BD Biosciences, USA) with CellQuest software (BD Biosciences, USA) used for analysis of the results.

Flp-In T-REx293 cells were seeded in 6 well plates at 50–60% confluence. After 24 hrs, doxorubicin (1 uM) and doxycycline (1 ug/ml) were added to each well. After 20 hours, cells were harvested and stained according to the protocol of FITC-Annexin V Apoptosis Detection kit (BD Pharmingen). Briefly, trypsinized cells were washed twice with cold PBS and then resuspended in 1X binding buffer at a concentration of 1 × 10⁶ cells/ml. 100 ul of the solution was transferred to 1.5 ml tube. 5 ul of FITC Annexin V and 5 ul PI were added to each tube and incubated for 15 mins at RT in the dark. Then 400 ul of 1X binding buffer was added to each tube. Stained cells were analyzed by using an Accuri Flow Cytometry (BD Biosciences). The percentage of FITC-positive cells was calculated using the CFlow software.

The internalization and distribution of cells were observed by a laser confocal fluorescence microscope. Briefly, SGC7901 cells were seeded in 35-mm cell petri dishes (Nest, 801002) and cultured 24 h for cell attachment. The cell density per dish was 5 × 10⁴. Next, replace the media with new serum-free media containing DSPE-PEG@ICG and DSPE-PEG-RGD@ICG, respectively (an equivalent ICG concentration: 1 mg/ml). After incubated for 4 and 12 h, the cells were washed with PBS and fixed with 4% paraformaldehyde. Afterward, the cells were stained with 10 μg/ml DAPI for 10 min and washed with PBS. Finally, a laser confocal fluorescence microscope (Zeiss LSM 710, Germany) was used for observing the binding and internalization of the micelles. The parameters were set at λ_ex 405 nm for the nuclei and λ_ex 633 nm for ICG.
For further quantitative analysis, 1 × 10⁵ SGC7901 cells were seeded in 12-well plates per well and cultured with serum-free media containing DSPE-PEG@ICG and DSPE-PEG-RGD@ICG (an equivalent ICG concentration: 1 mg/ml) for 12 h. At predetermined times, the cells were harvested and washed with PBS. Then, the cells were re-suspended in PBS and immediately quantitatively analyzed by Accuri flow cytometry (BD, United States).

Pancreatic cancer primary cells were seeded in six‐well plates at 50–60% confluence and harvested following treatment with 6‐TG (1 μm) for 48 h. After 48 h, cells were harvested and stained according to the protocol of the FITC‐Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). The percentage of apoptotic cell population was detected by Accuri Flow Cytometry (BD Biosciences) and calculated using the cflow software (National Institutes of Health, Bethesda, MD, USA).