Anti human cd34 pe

In this experimental study, MSCs serum-free medium
was ordered from Shanghai Pumao Biotechnology
(Shanghai, China), and trypsin and Dulbecco’s phosphate
buffered saline (DPBS) were ordered from Gibco (Grand
Island, NY, USA). The antibodies of APC-anti-human
CD73, FITC-anti-human CD90, PerCP-Cy5.5- antihuman CD105, PE-anti-human CD34, PE-anti-human
CD45, and PE-anti-human HLA-DR were purchased
from BD Pharmingen (NJ, USA). The CD63 and β-actin
antibodies were ordered from Absin Bioscience (Shanghai,
China) and Cell Signaling Technology (Danvers, MA,
USA), respectively.

For CD26⁺ and chemokine-like receptor-1⁺ (CMKLR1⁺) MSC sorting, MSCs were digested into single-cell suspensions and separately incubated with an anti-human CMKLR1 PE-conjugated antibody (R&D, USA) or anti-human CD26 FITC-conjugated antibody (BD, USA) for 30 min. After washing with phosphate-buffered saline (PBS) three times, CD26⁺ MSCs or CMKLR⁺ MSCs were isolated with a BD Influx cell sorter for later experiments according to the manufacturer’s instructions. MSCs incubated with PE- or FITC-conjugated IgG antibodies were used as negative controls.
To detect the positive rates of CD26 and CMKLR1 expression, MSCs were then incubated with an anti-human CMKLR1 PE-conjugated antibody (R&D, USA) and anti-human CD26 FITC-conjugated antibody (BD, USA) as described above and detected using a BD Influx cell sorter.
To detect cell markers of MSCs, MSCs were incubated with anti-human CD29 PE-conjugated, anti-human CD44 FITC-conjugated, anti-human CD105 FITC-conjugated, anti-human CD14 FITC-conjugated, anti-human CD34 PE, and anti-human CD45 PE-conjugated antibodies (all from BD Pharmingen, USA) as described above and then detected using a BD Influx cell sorter.

For phenotypic identification of the hDPSCs at P4, cells (1 × 10⁶) were digested with 0.25% (w/v) trypsin, washed twice with phosphate-buffered saline (PBS) and divided into aliquots. The cells were centrifuged, resuspended and stained with the following antibodies for 15 min at RT: anti-human CD34-PE, CD44-FITC, CD45-FITC, CD73-PE, CD90-FITC, and HLA-DR-FITC (BD Biosciences, USA). After washing, the cells were resuspended and then analyzed using flow cytometry instrument (FC500; Beckman Coulter, USA).

Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM-F12), penicillin/streptomycin and fetal bovine serum (FBS) were obtained from Gibco. Collagenase P were obtained from Roche. Anti-human CD34-PE, CD45-FITC, HLA-DR-FITC, CD44-FITC, CD73-PE, CD90-FITC, and were purchased from BD Biosciences. Adipogenic and osteogenic inducing medium from Cyagen. Matrigel were obtained from Corning. Trizol Reagent were purchased by Invitrogen. All-In-One RT MasterMix Kit, EvaGreen qPCR MasterMix Kit were obtained from abmGood. Cell-tracker dye CM-DiI were purchased from Invitrogen.

UCB samples were obtained from the Blood and Tissue Research Biobank (Barcelona, Spain). All samples were obtained between 20 and 72 h after delivery. To isolate CD34⁺ cells, UCB were incubated with RosetteSep (STEMCELL Technologies, Vancouver, Canada) for 20 min prior to density gradient purification using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) and centrifugation at 700g for 30 min. CD34⁺ cells were selected from the mononuclear cells fraction using the EasySep human cord blood CD34⁺ selection kit II (STEMCELL Technologies, Vancouver, Canada). Briefly, mononuclear cells were incubated with anti-human CD34 antibodies and FcR-blocking antibodies for 10 min. Then, dextran microbeads were added to the cell suspension and incubated for 1 min at room temperature. The immunomagnetic selection was performed using EasySep Magnets (STEMCELL Technologies) according to the manufacturer’s instructions. Purity, viability, and cell number were then assessed by flow cytometry using anti-human CD34-PE (BD Biosciences, San Jose, CA, USA), anti-human CD45-FITC (BD Biosciences), 7-amino-actinomycin-D (7-AAD, BD Biosciences) and Perfect Count Microbeads (Cytognos, Salamanca, Spain). Only samples with cell purity higher than 75% and viability higher than 90% were used.