Hla dr pe

Circulating neutrophils were identified by flow cytometry using the protocol described in our previous publications [19 (link), 54 ]. In brief, 30 μl of fresh blood were incubated with fluorochrome-labelled antibody cocktail (CD19-FITC, CD16-Pacific Blue, CD11b-APC (all from BD Biosciences, Oxford, UK) and HLA-DR-PE (eBioscience, UK)) for 45 min in the dark at 4 °C. Red blood cells were removed with lysis buffer (BD Biosciences) and samples were acquired using a BD Canto II flow cytometer (BD Biosciences). The flow cytometry data were analysed using FlowJo (version 10, Tree Star Inc., Ashland, OR, USA). Neutrophils were identified based on their cell size (FSC) and granularity (SSC) as well as their cell surface antigens (CD11b⁺CD16⁺HLA-DR⁻).

Freshly drawn blood samples (30 μl) were incubated with fluorochrome-labelled antibodies in a total volume of 100 μl FACS buffer (PBS/1% fetal calf serum (FCS)) for 45 min in the dark at 4 °C. Red blood cells were lysed by incubating the sample with 2 ml of 1X red blood cell lysis solution (BD Biosciences, Oxford, UK) for 10 min at room temperature (RT). After thorough washes samples were fixed in 1% paraformaldehyde (PFA) for 30 min in the dark at 4 °C. Samples were then washed and resuspended in PBS. All samples were examined by flow cytometry (FACS CANTO II; BD Biosciences), and data analysed blindly using the FlowJo software (version 10.07 for Windows, Tree Star, Ashland, OR, USA). The following anti-human antibodies were used for identification of leukocyte subsets: CD14-APC-Cy7, CD19-FITC, CD16-Pacific Blue, CD56-PE, CD8-PE-Cy7, CD4-Pacific Blue, CD11b-APC (all from BD Biosciences, UK) and HLA-DR-PE (eBioscience, UK). Live cells were gated for further analysis of leukocyte subsets. Different subsets of leukocytes were identified by relative cell size and granularity (FSC/SSC) or cell surface markers (Fig. 1). The neutrophil/lymphocyte ratio (NLR) was calculated by dividing the percentage of neutrophils by the percentage of lymphocytes.