Pkh67 green fluorescent cell linker kit

Exosomes were isolated from the atrial myocytes supernatant by ultracentrifugation. The cell culture supernatant was centrifuged at 300×g for 10 min, 2,000×g for 10 min, and 10,000×g for 30 min, followed by filtration through a 0.22 μm filter to eliminate cells, dead cells, and cellular debris. For exosomes purification, the supernatant was ultracentrifuged at 100,000×g for 70 min, followed by an additional washing step of the exosome pellet with PBS and centrifugation at 100,000×g for 70 min (Ultracentrifuge, Beckman Coulter, L8-70 M).
Exosomes were isolated and purified from serum using ExoQuick exosome precipitation solution (System Biosciences, EXOQ20A) according to the manufacturer’s instructions.
The protein content was measured using a BCA protein assay (Thermo Scientific). Atrial myocytes-derived exosomes were analyzed for the presence of the exosomal marker proteins ALIX, CD63 and CD81 using Western blot, and the relative expression levels of miR-210-3p and exosomal miR-210-3p were determined using qRT-PCR. For exosome uptake experiments, exosomes were observed and imaged using a Philips CM12 electron microscope (FEI Company) operated at 60–120 kV and equipped with a digital camera. Atrial myocyte-derived exosomes were labeled with the PKH67 Green Fluorescent Cell Linker Kit (Sigma, Aldrich) according to the manufacturer’s protocol.

Human THP1 monocytes stably transfected with a chimeric molecule consisting of the human FcγRIIIa-V158 extracellular domain joined to the transmembrane and intracellular domains of the gamma chain of the mast/basophil Fc receptor for IgE (THP1 CD16⁺ cell line) were used. The gamma chain allows FcγRIIIa membrane expression and provides the ITAM necessary for the transduction of the phagocytosis signal. THP1 CD16+ cells (1×10⁵) were pre-incubated with increasing concentrations (6.66×10^-5, 6.66×10^-6, 6.66×10^-7, and 6.66×10^-8 M) of 10% IVIg (Iqymune, LFB) or LFBD192 for 30 min at 37°C. Platelets were isolated from human blood (EFS, Les Ulis), labeled using the PKH67 Green Fluorescent Cell Linker Kit (general cell membrane labeling, Sigma), and sensitized at 37°C for 30 min with stirring with 1 µg/mL of a human IgG1 anti-CD41 monoclonal antibody (Absolute Antibody) or dilution buffer (non-sensitized platelet control). After incubation, samples were washed twice in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FCS at 1,200 × g for 5 min. Labeled platelets (2×10⁶) were added and incubated for 1 at 37°C. PKH67 labeling was quenched by trypan blue and effector cells were labeled with a fluorescent anti-CD45 antibody (Beckman Coulter). Antibody-mediated platelet phagocytosis was measured by flow cytometry (Gallios Flow Cytometer, Beckman Coulter).

HO1N1 cells were grown in 10 mm μ-Dishes (Ibidi GmbH) at a density of about 1 × 10⁴ cells per well. Prior to imaging, nuclear DNA was stained with NucBlue™ live cell stain according to the manufacturer’s protocol (Life Technologies, Grand Island, NY, USA). The cell membrane was labeled using PKH67 Green Fluorescent Cell Linker Kit (Sigma–Aldrich), according to the manufacturer’s instructions. Early endosome (EE) and late endosome (LE) were labeled using Invitrogen™/CellLight™ Early Endosomes-GFP, BacMam 2.0 (C10586, Invitrogen) and Invitrogen™/CellLight™ Late Endosomes-GFP, BacMam 2.0 (Invitrogen C10588, Invitrogen), following the manufacturer’s protocols. The staining solution was replaced with culture media and cells were immediately imaged. Confocal stacks were achieved with CLSM system (TCS SP8, Leica) using a 63 × 1.4 NA oil objective, and processing was completed using a 5% CO₂ incubator at 37 °C. Time-lapse images of the living cells were recorded every 5 s. Fiji software was used for quantification.^{64 (link)}

Purified microvesicles were labeled with the PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich, USA) according to the manufacture's protocol. The burn wounds on mice were injected with PBS or PKH67-labeled iPSCs-MVs immediately after burn. Fluorescence images were taken at days 1, 3 and 5 by the in vivo imaging system (Bruker, USA). The images were analyzed using Bruker MI SE 7.2 software.