Ficoll

This study was approved by the Institutional Animal Care and Use Committee and the Ethics Committee of Fudan University. Eight-week-old male Sprague-Dawley rats were used for EPC isolation. Bone marrow was isolated from the femur and tibia of the rats and then subjected to density gradient centrifugation using Ficoll (Sigma-Aldrich, #10771). Then, mononuclear cells were cultured on collagen I-coated dishes with EGM-2 medium (Lonza, #CC-3202) at 37°C in a 5% CO₂ incubator. After 3 days of incubation, the medium was changed every 3 days. When the cells had reached the second generation, the cells were stained with Dil (Sigma-Aldrich, #42364) and UEA (Sigma-Aldrich, #L9006). Laser confocal microscopy was used for observation of Dil and UEA staining.
The mononuclear cells in the peripheral blood were isolated by Ficoll density gradient centrifugation at 10, 20, or 30 days after the aneurysm model was established. CD34 (Abcam, #ab81289) and KDR (Vascular endothelial growth factor receptor-2, VEGFR-2) (Abcam, #ab9530) monoclonal antibodies were incubated with the mononuclear cells for 1 h on ice and then incubated with Alexa Fluor 546- (ThermoFisher, #A10036) and Alexa Fluor 488-(ThermoFisher, #A11008) labeled fluorescent secondary antibodies for 30 min. The proportion of CD34+/KDR+ cells was analyzed by flow cytometry (Beckman Coulter, DxFLEX).

As described previously^{32 (link)}, Ficoll (Sigma Aldrich, St. Louis, MO, USA), was added to EGM-Plus medium at a final concentration of 5% to mimic the viscosity of peripheral blood. To produce shear stress equivalent to that of physiological conditions in vivo (4–23 dyne/cm²), EGM-Plus media supplemented with Ficoll was perfused into the platform through a peristaltic pump. This produced a pressure waveform similar to that seen in capillary beds (2–5 mmHg). The pressure waveform is a function of the peristaltic pump’s roller configuration and rotor speed (rpm). Pump output is a high frequency, low amplitude wave that approximates the capillary bed frequency and pulse pressure. Pressure in the capillary and interstitial compartments reached equilibrium during the acclimation phase of incubation. The platform also has an O-ring to prevent leaks and completely separates the luminal and abluminal chambers. To maintain a stable microenvironment, gas-permeable reservoirs were used to exchange O₂ and CO₂.

Embryos were microinjected in a 3% Ficoll (w/v; Sigma-Aldrich, St. Louis, MO) solution in 1X MBS on plasticine-coated injection dishes. All embryos were seeded into Ficoll dishes 15 min before injection and allowed to heal for 1 hr in Ficoll after injection. All microinjections were performed at the four-cell stage.

Human peripheral T lymphocytes were isolated from whole blood of healthy adult donors by dextran sedimentation and Ficoll (Sigma) gradient separation followed by depletion of B cells using nylon wool column (Unisorb), to which B cells were adsorbed. Cells were incubated in a complete RPMI growth medium (500 ml RPMI Ca/Mg + heat inactivated FCS (10% final) + 5 ml 200 mM L-Glu (2 mM final) +5 ml 100 M Na pyruvate (1 mM final) + 5 ml Pen Strep antibiotics) for more than 2 h, and then non-adherent cells were harvested and transferred to a new plate, resulting in ~90% CD3+ T lymphocytes. Cells were then used for in vitro assay, in which 105 cells were plated per well in a 96-well plate and activated using CD3 and CD28 antibodies.

GFc7 nanocomplex was synthesized by Sodour Ahrar Shargh Company (Tehran, Iran). Minimum essential medium (α-MEM), penicillin G (100 U/ml), streptomycin (100 μg/ml), GlutaMAX, nonessential amino acids, trypsin-EDTA 0.25 %, and phosphate-buffered solution (PBS) were purchased from Gibco (Gibco-Life Technologies, Carlsbad,CA, USA). Hydrogen peroxide (H₂O₂), sodium isothiocyanate, dimethyl sulfoxide (DMSO), FeCl₃, nitric acid, acetone, methanol and formalin, Triton X-100, beta- glycerol phosphate, NHCl, and paraformaldehyde were purchased from Merck (Darmstadt, Germany). AB-human serum, propidium iodide (PI), hydrocortisone, isobutyl methyl xanthine, indomethacin, Oil Red stain, Alizarin Red stain, dexamethasone, ascorbic acid 2-phosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 99 % (MTT), p-nitrophenyl phosphate (pNP), Ficoll, and TRIzol were from Sigma-Aldrich (St Louis, MO, USA). IntraStain kit (Code-Nr.K2311) and all of the antibodies were obtained from Dako (Glostrup, Denmark) and Standard SYBR Green PCR kit from Fermentas, St. Leon-Rot, Germany.
The list of the equipment and instruments used is as follows: FACS Calibur (Becton Dickinson, Cockeysville, MD, USA), absorbance micro plate readers (ELx800™; BioTek, Winooski, VT, USA), Rotor Gene 6000 instrument (Corbett, Sydney, Australia), and scanning electron microscope VEGA-TESCAN-LMU model.

Islet isolation was performed as described by Schubert et al.34 (link). After euthanasia, a digestion solution containing Collagenase (Sigma-Aldrich, St. Louis, MO, USA) and DNase I (F. Hoffmann-La Roche, Basel, Switzerland) was injected anterogradely into the common bile duct. The pancreata were digested for 15 min., islets were isolated with a discontinuous Ficoll (Sigma-Aldrich) gradient and cultured in RPMI 1640 (Gibco, ThermoScientific, Venlo, Netherlands) supplemented with 10% (vol/vol) FBS (Biochrom). Purity and the amount of islets were determined using dithizone staining (Sigma-Aldrich).