Cd34 pe

Cell surface antigen phenotype was performed on cells at passage 3–6. Cells were detached using trypsin 0.2% solution and counted. Cells were liquated at 1–5 × 10⁵ cells in tubes, pelleted, and resuspended in 100 μL of phosphate buffered saline (PBS), pH 7.4, containing 0.5% bovine serum albumin (BSA). Then cells were stained with anti-human antibodies, CD90-fluorescein isothiocyanate (FITC) (eBioscience, USA), CD44-allophycocyanin (APC) (BioLegend, USA), CD45-V450 (BD Bioscience, USA), CD105- phycoerythrin (PE) (Santa Cruz Biotechnology, USA), CD34-PE (eBioscience, USA), and CD133-APC (Miltenyl Biotec, USA), at concentration of 1 μg/mL at room temperature for 1 h. The respective isotype antibodies were used as negative controls. The cells were pelleted, washed twice with PBS-BSA buffer, and resuspended in 100 μL buffer. Then, fluorescence-activated cell sorting (FACS) analysis was performed on BD Biosciences FACSAria flow cytometer (BD Biosciences, USA).

The expression of cellular markers, including a hematopoietic marker (CD34) and mesenchymal markers (CD105, CD73, and CD90), in ADSCs (passage 3) was examined using flow cytometry (CD90-FITC, CD73-APC, CD105-PECy7, and CD34-PE antibodies from eBioscience were used, San Diego, CA).
Moreover, the ability of ADSCs in differentiating into osteoblasts and adipocytes was assessed. ADSCs cultured in 6-well plates grew to approximately 50% confluence. Then, the ADSCs were cultured in osteogenic differentiation medium or adipogenic differentiation medium (Gibco, Carlsbad, CA) for 21 days. ADSCs were stained with alizarin red S and oil red O to verify the osteoblast and adipocyte differentiation of ADSCs, respectively.

10⁵ differentiating cells were harvested in phosphate‐buffered saline (PBS) containing 1% bovine serum albumin (BSA) (PBS/BSA) and centrifuged at 200 g for 5 minutes. Cell pellets were resuspended and mixed with the appropriate volume of antibody, CD34‐PE (12‐0349‐41, eBioscience, eBioscience Ltd., Hatfield, UK, http://www.ebioscience.com/), CD43‐APC (17‐0439‐42, eBioscience), CD235a‐FITC (11‐9987‐80, eBioscience), and CD71‐APC (17‐0719‐42, eBioscience), to a final volume of 100 μl PBS/BSA, incubated for 30 minutes then analyzed on a LSR Fortessa (BD Biosciences, Oxford, UK, http://www.bdbiosciences.com/) using FACS Diva. The proportion of enucleated cells present in the culture was assessed using CD235a‐FITC, CD71‐APC antibodies, LIVE/DEAD Fixable Near‐IR Stain (L10119, Thermo Fisher Scientific) and Hoechst dye (NucBlue, Thermo Fisher Scientific). Live CD235a⁺ cells were first gated, then anti‐CD71 and Hoeschst were used to define erythroblasts (CD71⁺/Hoechst⁺), nucleated RBCs (CD71⁻/Hoechst⁺) and enucleated RBCs (CD71⁻/Hoechst⁻) (Supporting Information Fig. S7).

Surface antigen expression on DPSCs was investigated as described previously^{47 (link)}. Briefly, after 2 × 10⁴ cells were reacted with CD29-PE, CD34-PE, CD45-PE, CD90-FITC, CD105-PE, CD146-FITC (eBioscience, San Diego, USA), and mouse IgG1 or IgG2a isotype control-PE (eBioscience), the samples were analyzed using an EC800 cell analyzer (Sony Biotechnology, Champaign, USA).

Anti-human CD66bFITC, CD3, CD19, CD14, CD163,CD138, CD56, CD15 all PE conjugated, HLADRV450, CD45V500, CD16APC-H7, CD14PE-Cy7, CD38PE-Cy7, CD 206APC were obtained (BD Pharmingen, Stockholm, Sweden). CD34PE, CD235aPE, CD11bPerCp-eFlour710 (eBioscience, San Diego, CA, USA), CD192APC, and CX3CR1AlexaFlour647 were from BioLegend, San Diego, CA, USA. Human FcReceptor binding inhibitor, 7AAD, and Annexin V-Alexa647, were obtained from eBioscience, San Diego, CA, USA, Sigma–Aldrich, (St. Louis, MO, USA), and Molecular Probes, Eugene, OR, USA, respectively.

Single cells obtained from E11 AGMs were dissociated by collagenase. Then antibody staining was performed for 30 mins at 4°C using antibodies specific to c-Kit-APC, CD31-PE, CD41-FITC, CD45-APC, CD34-PE and TER119-PE-CY7 (eBioscience). The cells were analyzed with the MoFlo XDP (Beckman Coulter).