{"id":2289,"date":"2017-06-08T20:53:52","date_gmt":"2017-06-09T00:53:52","guid":{"rendered":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/facs\/"},"modified":"2018-07-20T16:38:48","modified_gmt":"2018-07-20T20:38:48","slug":"facs","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/facs\/","title":{"rendered":"Preparation of yeast cells for FACS analysis"},"content":{"rendered":"<div>\n<p><em>A Dohlman Lab Protocol<\/em><\/p>\n<div>\n<p>1. Grow cells overnight to saturation.<\/p>\n<p>2. Dilute to A600nm=0.2 in the same media, grow to A600nm=1.0.<\/p>\n<p>3. Sonicate 1.5 ml for 10 sec, to disperse cells.<\/p>\n<p>4. Centrifuge 10 sec at maximum speed in a tabletop microfuge, and resuspend the pellet in 2 ml 70% EtOH, incubate overnight at 23 C with gentle shaking.<\/p>\n<p>5. Centrifuge as before, wash twice with 1 ml of 50 mM Tris-HCl pH 7.8, resuspend in 0.8 ml 50 mM Tris-HCl with 200 ug heat inactivated RNAseA, incubate at 37 C overnight.<\/p>\n<p>6. Centrifuge cells and resuspend in 0.5 ml of 50 mM Tris-HCl pH 7.8, 2.5 mg pepsin (powder) and incubate at 37 C for 30 min.<\/p>\n<p>7. Centrifuge and wash once with 1 ml 200 mM Tris-HCl pH 7.5, 211 mM NaCl, 78 mM MgCl2, and resuspend in 0.55 ml of the same buffer containing 50 ug propidium iodide (100X stock in water).<\/p>\n<p>Reference:<br \/>\nLi, F., Flanary, P. L., Altieri, D. C., and Dohlman, H. G., Cell division regulation by Bir1, a member of the IAP (inhibitor-of-apoptosis) family in yeast. J. Biol. Chem. 275:6707-6711, 2000.<\/p>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>A Dohlman Lab Protocol 1. Grow cells overnight to saturation. 2. Dilute to A600nm=0.2 in the same media, grow to A600nm=1.0. 3. Sonicate 1.5 ml for 10 sec, to disperse cells. 4. Centrifuge 10 sec at maximum speed in a tabletop microfuge, and resuspend the pellet in 2 ml 70% EtOH, incubate overnight at 23 &hellip; <a href=\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/facs\/\" aria-label=\"Read more about Preparation of yeast cells for FACS analysis\">Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":12,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2289","page","type-page","status-publish","hentry","odd"],"acf":[],"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2289","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/users\/22429"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/comments?post=2289"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2289\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2239"}],"wp:attachment":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/media?parent=2289"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}