{"id":278,"date":"2023-07-07T12:36:49","date_gmt":"2023-07-07T04:36:49","guid":{"rendered":"http:\/\/yuhongtaolab.com\/?post_type=resource&#038;p=278"},"modified":"2023-07-07T12:36:49","modified_gmt":"2023-07-07T04:36:49","slug":"esc-transfection-with-lipofectamine-stem-transfection-reagent","status":"publish","type":"resource","link":"http:\/\/yuhongtaolab.com\/en\/resource\/esc-transfection-with-lipofectamine-stem-transfection-reagent\/","title":{"rendered":"ESC transfection with Lipofectamine stem transfection reagent"},"content":{"rendered":"<p><strong>Forward transfection in 6-well plate<\/strong><br \/>\n1. Seed hESCS in 6-well plate and culture for 12-24 hours.<br \/>\n2. Prepare transfection mixture as follows:<br \/>\n1. Tube-1: 100ul Opti-MEM, 5ul Lipofectamine stem transfection reagent.<br \/>\n2. Tube-2: 100ul Opti-MEM, 2ug plasmid.<br \/>\n3. Mix tube-1 and tube-2, incubate at RT for 10min.<br \/>\n3. Change 1.5ml\/well fresh mTeSR1, add transfection mixture onto cells.<br \/>\n4. 24h after transfection, refeed 1ml fresh mTeSR1 without removing old medium.<br \/>\n5. 48h after transfection, passage cells to new 6-well plate, add 0.5ug\/ml puromycin and treat for 24h or until non-transfection control cells are all killed by puromycin.<br \/>\n6. Culture with mTeSR1 supplemented with Y27632 for 2 more days, then change to normal mTeSR1.<\/p>\n<p><strong>Reverse transfection in 6-well plate<\/strong><br \/>\n1. Prepare transfection mixture as follows:<br \/>\n1. Tube-1: 100ul Opti-MEM, 10ul Lipofectamine stem transfection reagent.<br \/>\n2. Tube-2: 100ul Opti-MEM, 2ug plasmid.<br \/>\n3. Mix tube-1 and tube-2, incubate at RT for 10min.<br \/>\n2. Treat cells with Versene or accutase, dissociate cells into singe cell suspension or small clumps (3-5 cells\/clump).<br \/>\n3. Seed cells into 6-well plate (5e5\/well or 1:5 split).<br \/>\n4. Add transfection mixture onto cells, culture in 37\u2103 incubator.<br \/>\n5. 24h after transfection, refeed 1ml more mTeSR1 without removing old medium.<br \/>\n6. 48h after transfection, passage cells into new 6-well plate, add 0.5ug\/ml puromycin and culture for 24h or until non-transfected control cells are all killed by puromycin.<br \/>\n7. Culture with mTeSR1 supplemented with Y27632 for 2 more days, then change to normal mTeSR1.<\/p>","protected":false},"featured_media":0,"template":"","class_list":["post-278","resource","type-resource","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/resource\/278","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/resource"}],"about":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/types\/resource"}],"wp:attachment":[{"href":"http:\/\/yuhongtaolab.com\/en\/wp-json\/wp\/v2\/media?parent=278"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}