Cd31 apc

Pancreatic tissue isolation was performed as previously^{36 (link)} with minor modifications. Briefly, pancreas was minced into small pieces, and transferred to 3 mL 0.5 mg/mL Collagenase P (Roche, 11213873001) dissolved in HBSS together with 5 mM glucose. Then the digestion medium was put into the water bath at 37 °C 5 min for acclimatization and gently shaking another 5 min for digestion. To stop the digestion, 10 mL DMEM containing 1% FBS was added to the medium. Tissues were pipetted up and down for further dissociation and filtered by the 70-μm filter. Then cells were centrifuged for 5 min at 1200 rpm and incubated with 1 mL red blood cell lysis buffer (eBioscience, 00-4333-57) at room temperature for 5 min. 10 mL cold PBS containing 1% FBS was added to stop digestion, followed by centrifugation. 1% Fc in PBS was added for 5 min to block, then the isolated cells were stained with fluorochrome-conjugated antibodies including CD45-APC (eBioscience, 17-0451-82, 1:400), CD31-APC (eBioscience, 17-0311-82, 1:40), CD140a-APC (eBioscience, 17-1401-81, 1:100), CD326-APC (eBioscience, 17-5791-82, 1:200) for 30 min at 4 °C. After washing with cold PBS, cells were resuspended in PBS containing DAPI (1:1000). Then flow cytometry experiments were performed using the Beckman Cytoflex LX or sorted using the Sony MA900 flow cytometer.

Retinas were harvested at P5, dissected in cold PBS and digested in 1 mg ml⁻¹ Collagenase type II (Sigma) in DMEM (Sigma). Cells were incubated with CD31-APC (BD 551262, 2 ng ml⁻¹) and CD45-FITC (11-0452-85, 5 ng ml⁻¹, eBioscience), Hoechst (B2261, 25 μg ml⁻¹, Sigma Aldrich) and Pyronin Y (P9172, 200 ng ml⁻¹, Sigma Aldrich). CD31+/CD45− endothelial cells were isolated by FACS and further analysed for their cell cycle distribution by two-dimensional analysis of Hoechst and Pyronin Y fluorescence signal.

The bone marrow of 14-day-old C57BL/6 mice was harvested and passed through a 40-μm cell strainer, yielding single cells. Subsequently, the single-cell suspension was incubated with antibodies specific for CD51-PE (1:200 dilution, BD, USA), CD45-FITC (1:200 dilution, eBioscience, USA), Ter119-PECY7 (1:200 dilution; eBioscience) and CD31-APC (dilution 1:200; eBioscience) at 4 °C for 30 min. CD51⁻CD45⁻Ter119⁻CD31⁻ cells (CD51⁻bMSCs) and CD51⁺CD45⁻Ter119⁻CD31⁻ cells (CD51⁺bMSCs) were sorted using flow cytometry (Influx, BD). Then, the isolated cells were allowed to adhere to culture plates (Corning, USA) in medium described as follows: DMEM/F12 (Gibco, USA) containing 10 ng/ml EGF (Pepro tech, USA), 10 ng/ml bFGF (Pepro tech), 2% B27 (Invitrogen, USA), 0.1 mM β-mercaptoethanol (Invitrogen), 1% l-glutamine (Sigma Aldrich, USA), 1% foetal bovine serum (Gibco) and 100 IU/ml penicillin/streptomycin (Invitrogen). Cells were cultured at 37 °C in a 5% CO₂ atmosphere and propagated every 2 or 3 days.

Dissociated cardiomyocytes were centrifuged and incubated with red-blood-cell lysis buffer (2–5 ml, eBioscience, 00-4333-57) for 5 min at room temperature. After washing with an equal volume of isolation buffer (2 mM EDTA and 0.25% BSA in PBS), cells were centrifuged at 20 g for 3 min. The re-suspended cells were first stained with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Life Technology, L34955) according to the manufacturer’s instruction. Then cells were washed with isolation buffer and centrifuge with 20 g for 3 min, and the pellets were blocked in Fc block (eBioscience, 14-0161, 1:100) for 5 min at room temperature and then directly incubated with antibodies mixture containing CD31 APC (eBioscience, 17-0311-80, 1:40), CD140a APC (eBioscience, 17-1401-81, 1:500), CD45 APC (eBioscience, 17-0451-82, 1:400) at 4 °C for 30 min. After washing with isolation buffer, cells were re-suspended with 500 µl isolation buffer and finally analyzed with FACS Aria II Flow Cytometer (BD Biosciences). The raw data were processed by FlowJo software (Tree star).

After isolation, remaining cells were incubated with anti-mouse CD16/CD32 (1:50, BD Pharmingen#553142) to block endogenous Fc for 10 minutes on ice. After this, cells were stained with antibodies including CD45-EF450 (1:2000, eBioscience #48-0451-82) and CD31-APC (1:100, eBioscience #17-0311-82) for 45 minutes at 4°C. After wash, the cells were resuspended in 500 μl buffer and analyzed on an LSR Fortessa (BD Pharmingen) cell analyzer. Obtained data were analyzed by Summit software (Beckman Coulter).