{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T09:45:15Z","timestamp":1775727915850,"version":"3.50.1"},"reference-count":9,"publisher":"Wiley","issue":"2","license":[{"start":{"date-parts":[[2024,2,7]],"date-time":"2024-02-07T00:00:00Z","timestamp":1707264000000},"content-version":"vor","delay-in-days":6,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2024,2,1]],"date-time":"2024-02-01T00:00:00Z","timestamp":1706745600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["RTG2727\u2013445549683"],"award-info":[{"award-number":["RTG2727\u2013445549683"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,2]]},"abstract":"Abstract<\/jats:title>\n Natural killer (NK) cells are innate cytokine\u2010producing 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\u2010regulate HIF\u20101\u03b1 (hypoxia inducible factor\u20101\u03b1), 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\u20101\u03b1 levels in primary human NK cells. One difficulty with this approach, however, is that NK cells are difficult\u2010to\u2010transfect cells and common transfection methods, including electroporation or lipofection, suffer from variable transfection efficiency and cell viability. Moreover, the detection of HIF\u20101\u03b1 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\u20101\u03b1 expression in primary human NK cells. We further provide a protocol for the detection of HIF\u20101\u03b1 by immunoblot analysis demonstrating its efficient downregulation by siRNA. \u00a9 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.<\/jats:p>\n \n Basic Protocol 1<\/jats:bold>\n : Isolation of natural killer cells from human peripheral blood mononuclear cells\n <\/jats:p>\n \n Basic Protocol 2<\/jats:bold>\n : Delivery of non\u2010coding small interfering RNA and HIF\u20101\u03b1 targeting siRNA into natural killer cells using ExPERT ATx\n <\/jats:p>\n \n Basic Protocol 3<\/jats:bold>\n : Assessing the downregulation of HIF\u20101\u03b1 protein using immunoblot analysis\n <\/jats:p>\n \n Support Protocol 1<\/jats:bold>\n : Exemplary assessment of transfection efficiency using fluorescently labeled non\u2010targeting siRNA\n <\/jats:p>\n \n Support Protocol 2<\/jats:bold>\n : Exemplary assessment of NK cell viability 20 hr post\u2010transfection\n <\/jats:p>\n \n Support Protocol 3<\/jats:bold>\n : Exemplary assessment of HIF\u20101\u03b1 knockdown using immunoblot analysis\n <\/jats:p>","DOI":"10.1002\/cpz1.987","type":"journal-article","created":{"date-parts":[[2024,2,8]],"date-time":"2024-02-08T02:00:00Z","timestamp":1707357600000},"source":"Crossref","is-referenced-by-count":1,"title":["Downregulation of Hypoxia Inducible Factor\u20101\u03b1 in Primary Human Natural Killer Cells Using Small Interfering RNA Delivery with ExPERT ATx by MaxCyte"],"prefix":"10.1002","volume":"4","author":[{"given":"Sonia Y.","family":"Vel\u00e1squez","sequence":"first","affiliation":[{"name":"Department of Anesthesiology and Surgical Intensive Care Medicine, Mannheim Institute of Innate Immunoscience (MI3) Heidelberg University Mannheim Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gizem E.","family":"Baslar","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology and Surgical Intensive Care Medicine, Mannheim Institute of Innate Immunoscience (MI3) Heidelberg University Mannheim Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jutta","family":"Schulte","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology and Surgical Intensive Care Medicine, Mannheim Institute of Innate Immunoscience (MI3) Heidelberg University Mannheim Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tanja","family":"Fuderer","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology and Surgical Intensive Care Medicine, Mannheim Institute of Innate Immunoscience (MI3) Heidelberg University Mannheim Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Holger A.","family":"Lindner","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology and Surgical Intensive Care Medicine, Mannheim Institute of Innate Immunoscience (MI3) Heidelberg University Mannheim Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5746-4496","authenticated-orcid":false,"given":"Anna","family":"Coulibaly","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology and Surgical Intensive Care Medicine, Mannheim Institute of Innate Immunoscience (MI3) Heidelberg University Mannheim Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,2,7]]},"reference":[{"key":"e_1_2_11_2_1_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2015.00266"},{"key":"e_1_2_11_2_2_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.02401"},{"key":"e_1_2_11_2_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010021\u201084916\u20100"},{"key":"e_1_2_11_2_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.micinf.2016.11.003"},{"key":"e_1_2_11_2_5_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.50.32253"},{"key":"e_1_2_11_2_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature06905"},{"key":"e_1_2_11_2_7_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMra1011165"},{"key":"e_1_2_11_2_8_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI84433"},{"key":"e_1_2_11_2_9_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.92.12.5510"}],"container-title":["Current 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