Pkh26 red fluorescent cell linker mini kit

Primers and phosphate-buffered saline (PBS) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Fe₃O₄@TiO₂ was obtained from Nanjing Xiuyuan Biotechnology Co., Ltd. (Jiangsu, China). Monoclonal anti-CD63 antibody, monoclonal calnexin antibody, and monoclonal TSG101 antibody were purchased from Abcam Co., Ltd. (Shanghai, China). A solution of 28 wt% ammonium hydroxide (NH₃•H₂O) and PKH26 Red Fluorescent Cell Linker Mini Kit were obtained from Sigma Co., Ltd. (Shanghai, China)., A 0.22-μm syringe-driven filter was purchased from Millipore (United States). Deionized water was processed by Ultrapure Millipore water (18.2 MΩ cm). High glucose Dulbecco’s modified Eagle’s medium (DMEM) and penicillin–streptomycin solution were purchased from Hyclone (UT, United States). Exosome-depleted fetal bovine serum (FBS) media supplement was purchased from System Biosciences, SBI (CA, United States).
All cell lines were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Healthy human and lung cancer patient plasma samples were supplied by Nanjing University-affiliated Drum Tower Hospital. The samples were centrifuged at 1,500 g for 10 min at 4°C. The supernatant was collected and centrifuged at 2,000 g for 10 min at 4°C to remove platelets. The supernatant was centrifuged at 15,000 g for 30 min and stored at −80°C.

When reached 90% confluences, the adherent cells were incubated in culture medium with 5% exosome-depleted fetal bovine serum (FBS) for 24 h, and 5–8 passages human umbilical cord MSC (hUCMSCs) were used for experiments. The conditioned medium was centrifuged at 4 °C at 300 g for 10 min at 2000 g for 10 min and finally at 10000 g for 30 min to remove the cells and debris, followed by centrifugation of the supernatant at 100000 g at 4 °C for 1 h, The hUCMSC derived exosomes (MSC-exo) were resuspended in PBS and filtered with a 0.22 μm microfiltration membrane, centrifuged again in PBS at 100,000 g for 1 h to collect the exosomes, which were re-suspended in PBS stored at − 80 °C. The protein concentration was determined using a bicinchoninic acid (BCA) assay kit, and labeled with PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma) according to the manufacturer’s protocol. MSC-exo The morphology of MSC-exo was examined using a transmission electron microscope (TEM) according to the manufacturer’s instructions. Briefly, the prepared exosomes were stained with phosphotungstic acid solution and then performed under a Hitachi-9000 TEM system. The expression of CD63, CD81, TS101 and Alix was confirmed by Western blot.

To evaluate whether CD8 T cells interacted with EVs, the PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma Aldrich, United States) was utilized to stain the membranes of EVs as per the manufacturer’s protocol. The experiment was performed based on the protocol by Ragni et al. (2017) (link). Briefly, 50 μg of isolated EVs was resuspended in 100 μl Diluent C. Separately, 1.4 μl PKH26 dye was mixed with 300 μl of Diluent C. The two components were combined and gently mixed, and incubated at room temperature for 5 min. The fluorescent labeling reaction was stopped by adding 700 μl of 1% FBS, and the stained EVs were co-cultured with 50,000 CD8 T cells in cell culture medium. Following incubation, cells were harvested and gently centrifuged at 10,000 × g for 5 min. The cells were then resuspended in 1 × PBS and analyzed by flow cytometry. EVs without PKH26 dye was incorporated into CD8 T cells as negative controls.

The protein in exosome was measured to quantify exosomes. The concentration of Exo was evaluated using the bicinchoninic acid (BCA, Thermo Fisher Scientific, Waltham, MA, USA). In Fig. 1f, hADSCs and hUCMSCs samples of whole-cell lysate (WCL) was reserved. The WCL were boiled with SDS–PAGE sample loading buffer, separated by SDS–PAGE, blotted on PVDF membranes. Western Blot with anti-CD9, anti-63 and anti-CD81 is used for identifying exosome. Transmission electron microscopy (TEM, FEI, USA) was used to examine the morphology of the isolated exosomes [51 (link)]. Nanosight tracking analysis (NTA, Malvern, USA) was performed to analyze exosome morphology. In order to analyze particle numbers, the Nanoparticle Tracking Analysis (NTA) 3.0 software was used. hADSC-Exo was labeled with PKH26 using the PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma, St Louis, MO, USA). Proteinase K used for chemical disruption of hADSC-Exo (Sigma, St Louis, MO, USA).

Tube formation experiments were performed with an Angiogenesis Starter Kit (Applied Biosystems) according to the manufacturer's manual. Human umbilical vein endothelial cells (HUVECs) seeded on the gel were cultured for 24 hours with the conditioned medium collected from the transfected HCT116 or HT29 cells. Cells were stained using a PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma‐Aldrich Inc) for microscopic visualization. Photographs were obtained using NIS‐Elements imaging software version 3.22.14 connected to the Eclipse Ti‐U inverted microscope system (Nikon Instruments Inc, Melville, NY). After overnight incubation at 37°C, the plates were photographed and the extent of tube formation was assessed.

To study the attachment and interactions of endothelial cells and smooth muscle cell sheet, the MSCORS-derived endothelial cells and MSCORS-SM-Sheet were pre-labeled with different fluorescent dyes. Briefly, the MSCORS endothelial cells obtained from endothelial differentiation were dissociated into single cells and labeled with an aliphatic fluorescent dye PKH26 (PKH26 Red Fluorescent Cell Linker Mini Kit, Sigma-Aldrich Chemie GmbH, Steinheim, Germany at a concentration of 2 × 10⁻⁶ M for 30 min in hypoxic conditions (5% O₂ and 5% CO₂ at 37 °C). The cells in MSCORS-SM-Sheet were labeled with PKH67 fluorescent dye (PKH67 Green Fluorescent Cell Linker Mini Kit Sigma-Aldrich Chemie GmbH, Steinheim, Germany) by directly incubating with PKH67 at 2 × 10⁻⁶ M in hypoxic conditions (5% O₂ and 5% CO₂ at 37 °C) for 2 h. The labeled endothelial cells were seeded directly onto the MSCORS-SM-Sheet. After 24 h of cultivation, the membrane complex was examined and imaged by LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

Huh-7 cells (a human HCC cell line) were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Ibaraki Osaka, Japan). HepG2 cells (a human HCC cell line) and HUVECs were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). LM3 cells (a mouse breast cancer cell line) were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China). GFP⁺ Huh-7 cells were generated by stably transfecting Huh-7 with GFP-encoding plasmids (GenePharma, Shanghai, China). SMAD3^−/− Huh-7 cells were generated by Crispr/Cas9 genome editing. FLAG-SMAD3 Huh-7 was generated by stably transfected with lentiviruses containing a FLAG-SMAD3 encoding sequence (Obio Technology, Shanghai, China). Huh-7, HepG2, and LM3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). HUVECs were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco). Both media were supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). For hypoxia treatment, cells were cultured in a hypoxic incubator with 1% O₂ for 24 h.
Diamide, NAC, and PKH26 Red Fluorescent Cell Linker Mini Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). The SMAD3 inhibitor SIS3 was purchased from Santa Cruz Biotechnology(CAS 1009104-85-1; CA, USA)