Quantibrite hla dr monocyte antibody cocktail

Expression of human leucocyte antigen-antigen D-related (HLA-DR) on CD14⁺ monocytes was measured by flow cytometry according to the manufacturer’s recommendations (Quantibrite HLA-DR/Monocyte antibody cocktail, BD Bioscience, Heidelberg, Germany). Briefly, 50 μL of EDTA-anticoagulated whole blood was incubated with 20 μl of the antibody cocktail and incubated for 30 min. Afterwards, erythrocyte lysis was performed by adding 450 μl FACS Lysing solution (BD Bioscience, Heidelberg, Germany) and further incubation for 15 minutes. Measurement was immediately done on a FACSCalibur flow cytometer (BD Bioscience). In addition, a 4-point calibration curve (Quantibrite PE Beads, BD Bioscience) was also measured daily to enable the transformation of the measured sample values to molecules of HLA-DR.

At each time point, samples were collected using EDTA tubes. All blood samples were stained within 1 h after collection or were immediately stored at 4 °C, in accordance with the standardization recommendations for mHLA-DR measurement [18 (link), 19 (link)]. For mHLA-DR measurement, staining and cell acquisition by flow cytometry (FC500, Beckman Coulter, Hialeah, FL, USA) were performed as described in the European standardized protocol and according to the manufacturer’s recommendations (Quantibrite HLA-DR/Monocyte antibody cocktail, BD Bioscience, San Diego, CA, USA). Results were expressed as the number of anti-HLA-DR antibodies per cell (AB/C) (normal >15,000), which is correlated with the number of HLA-DR molecules expressed on each monocyte. CD4+ T cells were measured by flow cytometry based on expression of CD4; Treg cells were identified based on expression of CD4, CD25, and CD127, and results were expressed as the total number of Treg cells and as the proportion of the total number of CD4+ T cells.