Anti cd8 apc

Fresh peripheral blood CD8⁺ T cells from healthy donors were enriched by negative selection using the RosetteSep Human CD8⁺ T Cells Enrichment Cocktail (StemCell Technologies) following manufacturer's protocol. Purified CD8⁺ T cells were stained with anti-CD8 APC and anti-HLA-DR PE antibodies (Immunotools) and sorted with a FACS Aria II^u flow cytometer (BD Biosciences), obtaining the following subsets: CD8⁺HLA-DR⁺ and CD8⁺HLA-DR⁻. Both subsets were collected into complete RPMI 1640 medium containing 50% FCS and washed before further experiments. The purity of each population, determined by flow cytometry, was always over 95%. For CD8⁺ cells depletion, peripheral blood mononuclear cells from healthy donors were isolated by density gradient, and CD8⁺ lymphocytes were depleted after staining with anti-CD8 APC (Immunotools). CD8⁺ depletion was ~99% effective.

Bronchoscopy with BAL was performed for diagnostic purposes according to the European Respiratory Society BAL Task Force Group guidelines [43 (link)]. Briefly, BAL was filtered through sterile gauze and centrifuged at 2000 rpm for 10 min at room temperature. The supernatant was then stored at −20 °C.
For the phenotyping of BAL samples, cells were stained with anti-CD3-FITC (Immunotools Friesoythe, Germany), anti-CD4-Viogreen (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD56-PE (Immunotools), anti-CD14-PE-Vio615 (Miltenyi Biotec), anti-CD45-PE-Cy7 (BD Biosciences, San Jose, USA), anti-CD8-APC (Immunotools), and anti-CD20-APC-Cy7 (Miltenyi Biotec). CD45⁺ cells were gated to select leukocytes. We identified CD4⁺ T cells (CD3⁺ CD4⁺ CD8⁻), CD8⁺ T cells (CD3⁺ CD4⁻CD8⁺), NK cells (CD3^-CD56⁺), and B cells (CD3⁻CD56⁻CD20⁺) on lymphocytes, gating leukocytes by size and complexity. Macrophages were identified by size, complexity, and CD14⁺ expression, and neutrophils were identified by size, complexity, and CD14⁻ as previously described in our laboratory [44 (link)]. Data acquisition and analyses were performed on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec) using FlowJo version 10 (FlowJo, Ashland, OR, USA).