Holger Lindner

Natural killer (NK) cells are innate cytokine‐producing and cytolytic effector lymphocytes. Their function is responsive to environmental factors, e.g., hypoxia, a frequent feature of inflamed tissues. Such responses require that the NK cells up‐regulate HIF‐1α (hypoxia inducible factor‐1α), the major mediator of cellular responses to hypoxia that affects cell survival as well as immune responses. Thus, a major approach to the study of NK cell effector function under hypoxic conditions involves the ability to regulate HIF‐1α levels in primary human NK cells. One difficulty with this approach, however, is that NK cells are difficult‐to‐transfect cells and common transfection methods, including electroporation or lipofection, suffer from variable transfection efficiency and cell viability. Moreover, the detection of HIF‐1α is technically challenging because of the rapid degradation of the protein under normoxic conditions. Here, using the commercially available ExPERT ATx by MaxCyte, we report a workflow for the reliable delivery of small interfering RNA (siRNA) for targeting HIF‐1α expression in primary human NK cells. We further provide a protocol for the detection of HIF‐1α by immunoblot analysis demonstrating its efficient downregulation by siRNA. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Isolation of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2 : Delivery of non‐coding small interfering RNA and HIF‐1α targeting siRNA into natural killer cells using ExPERT ATx Basic Protocol 3 : Assessing the downregulation of HIF‐1α protein using immunoblot analysis Support Protocol 1 : Exemplary assessment of transfection efficiency using fluorescently labeled non‐targeting siRNA Support Protocol 2 : Exemplary assessment of NK cell viability 20 hr post‐transfection Support Protocol 3 : Exemplary assessment of HIF‐1α knockdown using immunoblot analysis