{"id":947,"date":"2013-10-22T15:02:44","date_gmt":"2013-10-22T14:02:44","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=947"},"modified":"2014-11-26T11:30:03","modified_gmt":"2014-11-26T10:30:03","slug":"procyclic-form-culture","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/procyclic-form-culture\/","title":{"rendered":"Procyclic-form culture"},"content":{"rendered":"<p>SDM-79 powder is custom made by Life Technologies and is generally purchased on a consortium basis<\/p>\n<p>One pot is sufficient to make up 5 litres after addition of the following:<\/p>\n<table width=\"100%\" border=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td>Sodium bicarbonate<\/td>\n<td>10 g<\/td>\n<\/tr>\n<tr>\n<td>Pen-Strep (<a title=\"Pen-Strep product page\" href=\"http:\/\/www.lifetechnologies.com\/order\/catalog\/product\/15070063?ICID=search-product#\" target=\"_blank\">Life Technologies<\/a>)<\/td>\n<td>50 ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>Adjust to pH 7.4; make up to 4.5 litres with distilled water, filter sterilise, store at 4\u00b0C in 450 ml volumes.<\/p>\n<p>Prior to use, add 50 ml heat-inactivated fetal bovine serum (FBS; <a title=\"FBS product page\" href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/f7524?lang=en&amp;region=GB\" target=\"_blank\">Sigma<\/a>) and 1.5 ml hemin* (<a title=\"Hemin product page\" href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/h5533?lang=en&amp;region=GB\" target=\"_blank\">Sigma<\/a>); store at 4\u00b0C for up to two months.<\/p>\n<p>*Hemin stock solution \u2013 2.5 mg\/ml in 50 mM NaOH; filter sterilise and store as 20 ml aliquots at 4\u00b0C.<\/p>\n<p>Cultures should be grown in non-vented flasks at 28\u00b0C, and maintained at a density of 1&#215;10<sup>6<\/sup> to 2&#215;10<sup>7<\/sup> cells per ml.<\/p>\n<p><i>NB: growth at low density should be avoided unless in the presence of conditioned medium (grow a culture to ~1&#215;10<sup>7<\/sup> cells per ml centrifuge twice and filter sterilise through a 0.2 \u00b5m filter to remove all cells, and supplement with 10% heat-inactivated FBS; use within one week) or wild type cells under drug selection.<\/i><\/p>\n","protected":false},"excerpt":{"rendered":"<p>SDM-79 powder is custom made by Life Technologies and is generally purchased on a consortium&#8230;<\/p>\n","protected":false},"author":116,"featured_media":0,"parent":17,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-947","page","type-page","status-publish","hentry","odd"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.9 - 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