A Method of Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood.
Abstract
The majority of data on human Natural Killer (NK) cell phenotype and function has been generated using cryopreserved peripheral blood mononuclear cells (PBMCs). However, cryopreservation can have adverse effects on PBMCs. In contrast, investigating immune cells in whole blood can reduce the time, volume of blood required, and potential artefacts associated with manipulation of the cells. Whole blood collected from healthy donors and cancer patients was processed by three separate protocols that can be used independently or in parallel to assess extracellular receptors, intracellular signaling protein phosphorylation, and intracellular and extracellular cytokine production in human NK cells. To assess extracellular receptor expression, 200 μL of whole blood was incubated with an extracellular staining (ECS) mix and cells were subsequently fixed and RBCs lysed prior to analysis. The phosphorylation status of signaling proteins was assessed in 500 μL of whole blood following co-incubation with interleukin (IL)-2/12 and an ECS mix for 20 min prior to cell fixation, RBC lysis, and subsequent permeabilization for staining with an intracellular staining (ICS) mix. Cytokine production (IFNγ) was similarly assessed by incubating 1 mL of...Continue Reading
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