Computational protocol: Reconstituted High-Density Lipoprotein Modulates Activation of Human Leukocytes

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Protocol publication

[…] Heparinized whole blood from healthy volunteers was collected into pyrogen-free tubes, to which 5 μg/ml phytohemagglutinin-M (PHA, Calbiochem, Massachusetts, USA) was added for leukocyte stimulation. Simultaneously, rHDL was added to the whole blood at concentrations ranging from 0.04 to 1 mg/ml and incubated overnight at 37°C, 5% CO2 in a humidified atmosphere. Addition of substances resulted in a 1:2 dilution of human whole blood. The following day all manipulations were performed at 4°C or on ice. The cells were directly stained with antibodies specific for CD14, ICAM-1 and CD45 (BD Biosciences) for 30 min on ice. Red blood cells (RBC) were lysed by a 30 min incubation with EC Lysis Buffer (Qiagen AG, Basel, Switzerland) and gentle mixing. With the majority of RBC lysed, the tubes were centrifuged (400×g, 10 min, 4°C) and the cells resuspended in 300 μl PBS. Data acquisition and analysis was performed on a FACSCanto II flow cytometer employing the BD FACSDiva software (both BD Biosciences AG).For analysis, leukocytes were identified by gating on the pan-leukocyte surface marker CD45 which enables to exclude remaining non-lysed RBC from analysis. Then granulocytes were separated using granularity (side scatter; SSC) and monocytes using CD14 as marker which is expressed on the majority of monocytes (>90% of the whole population). This procedure allowed the assessment of ICAM-1 expression on the cell surface of each of these cell populations (all antibodies from BD Biosciences). To compare the levels of up-regulation of the indicated surface molecules, the median fluorescence intensity (MFI) ratios were calculated by dividing the median fluorescence of PHA-treated gated cells populations i.e. granulocytes and CD14+ monocytes by the median fluorescence of non-treated cells (indicated as fold increase MFI). [...] Cells were incubated with FITC-labeled monoclonal antibody (mAb) against CD80, CD83 and CD86 (BD, Franklin Lakes, NJ, USA) or Isotype Control IgG1 (BD).For determination of viability, propidium iodide (PI; Invitrogen; 5 μg/ml) was added to stained cells immediately before analysis by flow cytometry. As control for cell viability staining, cells were treated with PBS containing 0.1% BSA (Sigma) and 0.1% saponin (Sigma) and then stained with PI. To compare the levels of up-regulation of the indicated surface molecules, the median fluorescence intensity (MFI) ratios were calculated by dividing the median fluorescence of TLR-treated MoDC by the median fluorescence of non-treated MoDC (indicated as fold increase MFI). Measurements were performed with a BD FACScan flow cytometer and the obtained data were analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Homo sapiens
Diseases Atherosclerosis