Cfda se

The GC-2spd cell line was purchased from American type culture collection (ATCC, CRL-2196) and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C, 5% CO₂. Cells were labeled with Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; C0051, Beyotime, Shanghai, China) and then plated onto 6-well plates at a density of 1×10⁵ cells per well. PM_2.5 stock solution (10 mg/mL) was diluted to the final concentration of 12.5, 25, 50, 100, 200 μg/mL (0, 3.91, 7.81, 15.625, 31.25, 62.5 μg/cm²) with complete medium. Then, cells were treated with different concentrations of PM_2.5 suspensions. Meanwhile, the control group was added with the normal culture medium and PBS. The final concentration of PBS was less than 1%. After 24 h or 48 h, the cells were harvested, washed with PBS, and resuspended in HBSS. The fluorescence intensity was measured by a BD Accuri™ C6 flow cytometer (BD Pharmingen, San Diego, CA, United States) at an excitation wavelength of 488 nm. The FL1 detection channel was used to perform analysis, and 10,000 events were collected for each sample.

For cellular tracking after transplantation, cells were labeled with carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE, Beyotime, Shanghai, China). MSCs were resuspended in 1 mM CFDA dye at 37 °C for 20 min. The animals received stereotaxic transplantation within 15 min after reperfusion via a 10-ml Hamilton syringe (Hamilton, Bonaduz, Switzerland) injection. The transplant coordinate was 2 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture and 3 mm under the dura which is in peri-ischemic area. We injected MSC suspension with 1 × 10⁵ cells/5 μl potassium phosphate (PBS) buffer at a rate of 0.5 μl/min.
As a positive control, 2-[[(4-phenoxyphenyl) sulfonyl]methyl]-thiirane (SB-3CT; Sigma), an inhibitor of MMP-9, was also used. It was diluted in 10% dimethylsulfoxide/ 90% NS and was injected intraperitoneally at 10 mg/kg per day for 3 consecutive days beginning from days 1 to 3 after ischemia.

EPCs were pre-treated with IFN-γ (10 ng/ml, 48 h) or left untreated. A total of 5×10⁴ cells/well stimulator cells (pre-treated EPCs; un-treated EPCs; MSCs; pre-treated EPCs and MSCs (2:3); un-treated EPCs and MSCs (2:3)) were washed and seeded into a 96-well plate separately. For mixed lymphocyte reactions (MLR), all the attached stimulator cells were pre-treated with 10 μg/ml mitomycin C for 2 h.
Human PBMCs were collected from healthy individuals who gave informed consent. Allo-PBMCs were isolated from human peripheral blood by density gradient centrifugation using Ficoll. Isolated PBMCs were incubated with red blood cell lysis buffer (Qiagen, Germany) for 5 min at room temperature and washed twice with DPBS. A total of 2×10⁵ PBMCs were labeled with CFDA-SE (2 μM, 5 min; Beyotime), added to each well and co-cultured with different stimulator cells. All cells were cultured in RPMI 1640 (HyClone, USA) with 10% FBS and 2 μM L-glutamine (Sigma, USA). PBMCs cultured alone or stimulated by mitogen phytohaemagglutinin (PHA, 5 μg/ml; Sigma, Australia) acted as a negative control and positive control, respectively. After one week of co-culture, suspended PBMCs were harvested; proliferation of lymphocyte and T lymphocyte subsets were analyzed by flow cytometry.

PKH-26 kit (Sigma, USA, PKH26GL-1KT) was used to label exosomes according to the manufacturer’s protocol. The microtia chondrocytes were labelled with carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (Beyotime, China, C1031) according to the manufacturer’s protocol. The labelled exosomes were added into the culture dish or mixed together with Gelma hydrogel and microtia chondrocytes for 24 h at the same culture dish or hydrogel. The details of labelling could be seen at Additional file 1: 1.3. We used the confocal microscopy imaging to observe the uptake of exosomes for the 2D or 3D culture.

Anti-CD80 monoclonal antibody (66,406-1-Ig, Proteintech), anti-CD163 monoclonal antibody (GB13340, Servicebio), anti-CD8 monoclonal antibody (GB12068, Servicebio), anti-CD31 monoclonal antibody (GB113151, Servicebio), VEGFA Monoclonal antibody (19,003-1-AP, Proteintech), anti-CD47 monoclonal antibody (ab218810, Abcam), carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (C0051, Beyotime), PerCP anti-CD68 (333,813, BioLegend), PE anti-CD11b (101,208, BioLegend), granulocyte–macrophage colony-stimulating factor (GM-CSF) (C003, novoprotein), FITC-labeled anti-CD47 (CC2C6, BioLegend), Human Lymphocyte separation medium (7,111,011, DAKEWE). The fusion protein SIRPα-Fc has been engineered based on the initial extracellular domain of SIRPα and is currently undergoing Phase I/II clinical trial (NCT05140811) [32 (link)]. SIRPα-VEGFR1 is constructed by combining the extracellular domain of SIRPα with that of VEGFR1 (GenBank accession number: MG920788).

E. durans KLDS6.0930 at 2 × 10⁸ CFU/mL was fluorescently labeled with carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) (Beyotime, China) according to the manufacturer's instructions. Male rats were randomly divided in two groups of six rats each. The control group received 1 mL normal saline. The experimental group was treated with 2 × 10⁸ CFU E. durans KLDS6.0930 labeled with cFDA-SE at by gavage in 1 mL of sterile normal saline. After administration for 1 day, test rats were anesthetized with diethyl ether and sacrificed for cecum tissue collection. Cecal contents were carefully removed, and the cecal wall was rinsed with 1 mL sterile normal saline. Samples were stored in the dark and subjected to flow cytometry (BD LSR Fortessa) using a 488 nm laser.