Flow-Cytometric Immune Function Methodology(2)

42 Willmannfunction of fresh peripheral blood DCs, eliminating extensive leukocyte processing and enrichment procedures (1,2,4). The described protocols in Subheading 3. give examples of tumor necrosis factor (TNF)-α, interleukin (IL)-1-β, and IL-6 cytokines and their kinetics, as well as the detection of CD80 and CD83 well-established DC surface maturation and activation markers (17,23,38).

The DC method is composed of four basic steps: the activation of whole peripheral blood (Subheading 3.1.), sample processing (Subheading 3.2.),flow-cytometric sample acquisition (Subheading 3.3.), and data analysis(Subheading 3.4.). During cell activation, blood is exposed to an activating agent, in this case bacterial lipopolysaccharide (LPS). To detect intracellular cytokines, the simultaneous addition of a secretion inhibitor is crucial. BrefeldinA (BFA) is used to disrupt the Golgi transport, which leads to intracellular accumulation of otherwise secreted cytokines. When surface antigens are chosen as a functional readout, Golgi-disruptive agents cannot be used because they also block the transport of newly synthesized integral proteins to the cell surface. Therefore, two different protocols apply for the detection of surface versus intracellular targets as functional readouts (Subheadings 3.1.1. and 3.1.3.). The sample processing requires 2 h and is the most labor-intensive step of the procedure. The aliquoted blood volume can vary from 100 to 300 μL using the described 5-mL tube format. The author recommends the use of 300 μL blood, not older than 24 h. The sample processing step includes, in chronological order, cell surface staining, red blood cell lysis, cell permeabilization, and intracellular cytokine staining (Subheading 3.2.1.) or cell surface staining and red blood cell lysis for surface functional readouts (Subheading 3.2.2.). Washing steps are conducted after surface staining, permeabilization, and intracellular staining. Cell xation takes place during the red cell lysis and cell permeabilization. The processed sample should be acquired within 1 h on a ow cytometer. Standard ow-cytometric procedures use two scatter parameters, representing cell size and cell granularity, and up to four their cellular function, by changes in immunophenotype and protein secretion pattern, respectively. Here, the data analysis requires the combination of three uorescence parameters to allow the identi cation of CD11c+ and CD11c–peripheral blood DCs, leaving one uorescence parameter to determine the functional readout per sample. The time-to-answer of the methodology varies depending on the duration of sample activation. For TNF-α, the time is 4 h:2 h for cell activation and 2 h for sample processing. The basics of the procedure can be applied to other intracellular targets, activation agents, and different subsets of DC populations. To address higher-throughput needs, it is possible to scale down the assay to a 96-microtiter-well plate format.