Hla dr antibody

The characteristics of MSCs (p6) isolated from ten independent donors were analyzed by flow cytometry. MSCs were collected and labeled with fluorescein isothiocyanate (FITC)-conjugated human CD14, CD45, and human leukocyte antigen (HLA)-DR antibodies (BD Biosciences, Franklin Lakes, NJ, USA). Phycoerythrin (PE)-conjugated human CD73, CD166 (BD Biosciences), CD90, and CD105 (Invitrogen, Carlsbad, CA, USA) antibodies were used to measure stem cell surface markers on MSCs. An isotype control was also included. Washed MSCs were fixed with 1% (v/v) paraformaldehyde (Sigma-Aldrich). Stained cells were analyzed by flow cytometry on a MACSQuant instrument (Miltenvi Biotec, Bergisch Gladbach, Germany).

HUCMSC were isolated and characterized as previously described (9 (link)). Briefly, umbilical cord tissues from healthy women were washed three times and cut into approximately 1 mm³ pieces. The pieces evenly spread on the bottom of a 75cm² flask with the serum-free MSC culture medium (TBD Science, China) for primary adherent culture and cultured in a 5% CO₂ incubator at 37°C. The medium was refreshed every 3-4 days. After achieving 80%–90% confluence, the cells were digested with 0.25% trypsin-EDTA (Gibco, Carlsbad, CA, USA) 2 minutes for passaging. The cells were seeded into 175cm² flasks at a density of 6000-8000 cells/cm². Until the cell confluence reached 80%-90%, the cells were digested and collected to obtain the passage one of HUCMSC. Repeat the above operations for subculture. HUCMSC at passage five were characterized by morphology, mesenchymal lineage differentiation and surface marker expression. The capacity of osteogenic, adipogenic, and chondrogenic mesenchymal lineage differentiation was detected using Alizarin Red, Oil Red O, and Alcian Blue staining (OriCell, Guangzhou, China), respectively. Surface marker expression was also characterized by flow cytometry with CD73, CD90, CD105, CD34, CD14 and HLA-DR antibodies (BD Bioscience, San Jose, CA, USA) as described by previous study (20 (link)). Then the cells were used for further experiments.

PBMCs were cultured in RPMI 1640 medium (Capricorn) supplemented with 10% AB human serum (Sigma), 1% sodium pyruvate 100 mM (Gibco), 1% nonessential amino acids (Gibco), 1% HEPES buffer (Gibco), 0.5% of β-mercaptoethanol 10⁻² M (Gibco), and 0.2% of 10 mg/ml gentamicin (Gibco).
The recombinant form of PpSP32 was produced as described previously [22 (link)]. The following monoclonal antibodies were used for flow cytometry analysis: fluorescein isothiocyanate (FITC), allophycocyanin (APC), and phycoerythrin (PE) conjugated with anti-cluster of differentiation (CD)14, anti-CD86, and human leukocyte antigen receptor (HLA-DR) antibodies, respectively, (BD Biosciences).