Hla abc pe

ASCs were further expanded in vitro and were analyzed at passages 4 to 5 by flow cytometry (FACSAria; BD Biosciences, Erembodegem, Belgium). Monoclonal antibodies against CD9‐PE, CD10‐PECy7, CD13‐PE, CD14‐PECy, CD19‐PECy7, CD29‐APC, CD49d‐PE, CD73‐PE, CD90‐APC, CD106‐PE‐Cy5, CD146‐PE, and CD166‐PE (BD Biosciences); CD45‐FITC (Miltenyi Biotech, Bergisch Gladbach, Germany); CD31‐FITC, CD34‐APC, CD44‐FITC, HLA‐ABC‐PE, and HLA‐DR‐PE (Immunotools GmbH, Friesoythe, Germany); and CD105‐PE (R&D Systems Inc., Minneapolis, Minnesota) were used. Analysis was performed on 10,000 cells per sample. The positive expression was defined as the level of fluorescence greater than 99% of the corresponding unstained cell sample.

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.