Pe cyanine7 anti mouse cd80

Female C57BL/6 mice (6–8 weeks) were purchased from Shanghai SLAC Co. Ltd. All in vivo experiments were performed according to protocols approved by the Laboratory Animal Center of The Second affiliated Hospital of Zhejiang University School of Medicine. Mice were randomly divided into six groups (n = 5), including: (1) PBS, (2) PBS + US (3) PPIX NDs, (4) PPIX ND + US, (5) PMPS NDs and (6) PMPS NDs + US. PanC02 cells ( ${2\times 10}^6$ ) were orthotopically injected into the pancreas of each mouse. Seven days later, the tumors were allowed to reach 50 ~ 100 mm³ before the experiments. The mice in other groups were administered with PBS, PPIX NDs (PPIX 10 mg/kg), or PMPS NDs (equal dose of PPIX:10 mg/kg). In US irradiation group, US (Frequency: 3 MHz; Acoustic intensity: 1.0 W/cm²; Duty cycle: 20%; Times: 5 min) was conducted at five minutes and 6 h post-injection. On the third day, the tumor-draining lymph nodes were excised for analysis by FCM after co-staining with anti-mouse CD11c BV421 (Biolegend, 117329), anti-mouse CD80 PE/cyanine7 (Biolegend, 104733), and anti-mouse CD86 BV785 (Biolegend, 105043). Meanwhile, the blood samples were collected at 72 h after treatments, and the proinflammatory cytokines, including IL-6 and TNF-α, were tested by ELISA.

According to an established method, Bone-marrow-derived DCs (BMDCs) were isolated from 6–8-week C57BL/6 mice. The PanC02 cells after different treatments (PBS, PBS + US, PPIX NDs, PPIX NDs + US, PMPS NDs, PMPS NDs + US) were incubated with BMDCs respectively. After 2-day stimulation, the BMDCs were DCs stained with anti-mouse CD11c BV421 (Biolegend, 117329), anti-mouse CD80 PE/cyanine7 (Biolegend, 104733), and anti-mouse CD86 BV785 (Biolegend, 105043) were analyzed by FCM (Beckman CytoFlex).

Regarding the stromal vascular component (SVF) isolation from adipose tissues, tissues were cut with scissors and digested with collagenase (14065056, Gibco, New York, NY, USA) in HBSS (14065056, Gibco, New York, NY, USA) in a 37 °C shaking water bath. Cells from spleen were obtained by direct grinding spleen tissue without enzymatic digestion and passed through cell filter and centrifuged before resuspension in red cell lysis buffer prior to further analysis.
For FACS analysis, after red blood cell lysis, Cells were collected at 4 °C and then resuspended in block buffer with 0.2%BSA in PBS and Anti-mouse CD16/32 (TruStain FcX, Biolegend, San Diego, CA, USA) for 0.5 h. Subsequently, cells were stained with surface antibodies at 4 °C for 30 min. Following antibodies were all purchased from Biolegend: PE anti-mouse F4/80, PE/Cyanine7 anti-mouse CD80, Alexa Fluor anti-mouse CD206, APC anti-mouse CD45, PE anti-PPmouse CD140a (PDGF Receptor a). Immune cells were firstly isolated using mouse CD45+ nanobeads (Biolegend, San Diego, CA, USA). Flow cytometry data were collected using Fortessa (BD LSR Fortessa, Franklin Lakes, NJ, USA). FACS analysis were carried out using FlowJo-V10 (FlowJo LLC, Ashland, OR, USA).