Hla dr pe

To characterize hAF-MSCs, the cell surface markers of cells cultured in basic media containing 10% FBS or 10% hPL were evaluated. The cells were trypsinized with 0.25% trypsin-EDTA at 37°C for 1 min and centrifuged at 2,035 × g for 6 min at room temperature to obtain the cell pellets. Then, non-specific binding was blocked using 10% human AB serum [serum from type AB donors; processed by our laboratory and inactivated at 53°C for 90 min as previously described (16 (link))] at 4°C for 30 min. Subsequently, they were incubated with the following monoclonal antibodies: Mouse anti-human CD34-FITC (cat. no. 343504; BioLegend, Inc.), CD44-FITC (cat. no. 21810443; ImmunoTools GmbH), CD45-phycoerythrin (PE; cat. no. 304008; BioLegend, Inc.), CD73-PE (cat. no. 21270734; ImmunoTools GmbH), HLA-ABC-FITC (cat. no. 21159033; ImmunoTools GmbH) and HLA-DR-PE (cat. no. 21388994; ImmunoTools GmbH). Cell surface marker expression was detected using a FACScan (BD Biosciences) and analyzed using CellQuest™ Pro 9.0 software (BD Biosciences).

Immature DCs were treated with different amounts of NLC, diluted from formulations described in Table S1, in presence or absence of 100 ng/mL of TNF-α (Immunotools), and allowed to mature for 72 hours, before being harvested. Flow cytometry was performed as previously described.25 (link) Briefly, cells were washed and labeled (for 45 minutes at 4°C in the dark) in staining buffer with combinations of the following antihuman antibodies: CD1a, CD11c, CD86-FITC, CD40-FITC, HLA-ABC-PE, human leukocyte antigen – antigen D related (HLA-DR-PE) (all from Immunotools), CD83-FITC (AbDSerotec, Kidlington, UK). Isotype and fluorochrome-matched control antibodies were used to define background staining. For cell viability, Annexin V-FITC staining was performed for 15 minutes, followed by propidium iodide (PI; BD Biosciences, San Jose, CA, USA) staining, just prior to acquisition. For each sample, 10,000 cells were acquired, gated according to forward and side scatter parameters. Samples were analyzed using a FACS Calibur flow cytometer with Cell Quest software, or FACS Canto II flow cytometer, with Diva software (all from BD Biosciences, Franklin Lakes, New Jersey, USA). Results were analyzed using FlowJo software (TreeStar, Inc., Ashland, OR, USA). The mean fluorescence intensity for each sample was calculated subtracting the mean fluorescence intensity of the respective isotype control.