Downregulation of Hypoxia Inducible Factor-1α in Primary Human Natural Killer Cells Using Small Interfering RNA Delivery with ExPERT ATx by MaxCyte.

Abstract

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.