Anti hla dr anti monocyte quantibrite assay

mHLA-DR expression, in numbers of antibodies bound per monocyte (AB/cell), was analysed at three time points (day 1 or 2, day 3 or 4 and day 6, 7 or 8) using the Anti-HLA-DR/Anti-Monocyte Quantibrite assay (BD Biosciences, San Jose, USA), as detailed in the Supplementary Material.

T lymphocyte subpopulation immunophenotyping was performed on an automated volumetric flow cytometer from Beckman Coulter (Aquios CL) as previously described [19 (link)]. Monocyte HLA-DR expression and B and NK immunophenotyping were performed using antibodies from Beckman-Coulter and BD Biosciences. The expression of monocyte HLA-DR was determined using the Anti-HLA-DR/Anti-Monocyte Quantibrite assay (BD Biosciences, San Jose, USA). A total number of antibodies bound per cell (AB/C) were quantified using calibration with a standard curve determined with BD Quantibrite phycoerythrin (PE) beads (BD Biosciences) as described elsewhere [20 (link)]. B and NK lymphocyte immunophenotyping was performed using lyophilized antibody panel from Beckman Coulter (Duraclone kit). Data were acquired on a Navios flow cytometer (Beckman Coulter, Hialeah, FL), and flow data were analyzed using Navios software (Beckman Coulter). Enumeration of lymphocyte subpopulations as well as mHLA-DR measurement were performed using standardized protocols fulfilling clinical and diagnostic laboratories accreditation requirements from the International Organization for Standardization.

Monocyte HLA-DR (mHLA-DR) expression was assessed in ethylenediaminetetraacetic-acid (EDTA)-anticoagulated whole blood using the Anti-HLA-DR/Anti-Monocyte Quantibrite assay (BD Biosciences, San Jose, California, USA) on a CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, Indiana, USA). Flow data were analyzed using Kaluza Software (Beckman Coulter, Indianapolis, Indiana, USA). The number of antibodies bound per cell was calculated by standardizing the geometric mean of monocyte HLA-DR fluorescence intensity (MFI) to BD Quantibrite phycoerythrin (PE) beads (BD Biosciences, San Jose, California, USA). mHLA-DR expression was assessed on both LPS challenge days one hour before, as well as three and six hours after LPS administration.