{"id":905,"date":"2013-09-16T22:20:38","date_gmt":"2013-09-16T21:20:38","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=905"},"modified":"2014-11-26T11:30:03","modified_gmt":"2014-11-26T10:30:03","slug":"bsf-to-procyclic-differentiation","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/bsf-to-procyclic-differentiation\/","title":{"rendered":"BSF to procyclic differentiation"},"content":{"rendered":"<p>Adapted from Overath <em>et al<\/em>\u00a0 (1986) <a title=\"PubMed\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/3769918\" target=\"_blank\"><em>European Journal of Biochemistry<\/em> <strong>160<\/strong>:175<\/a><\/p>\n<p><em><strong>Materials<\/strong><\/em><\/p>\n<ul>\n<li>10xDTM (store in 50 ml aliquots at -20\u00b0C) &#8211; see bottom of page for recipe<\/li>\n<li>Hemin (<a title=\"Hemin product page\" href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/h5533?lang=en&amp;region=GB\" target=\"_blank\">Sigma<\/a>; 2.5 mg\/ml in 50 mM NaOH; sterile filter and store at 4\u00b0C)<\/li>\n<li>100x CCA (300 mM sodium citrate, 300 mM cis-aconitate; sterile filter and store at -20\u00b0C)<\/li>\n<\/ul>\n<p><strong><em>Protocol<\/em><\/strong><\/p>\n<ul>\n<li>Prepare 500 ml DTM as follows and store at 4\u00b0C for up to 3 months<\/li>\n<\/ul>\n<table width=\"100%\" border=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td><\/td>\n<td><span style=\"font-family: arial\">grams unless stated<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">10xDTM<\/span><\/td>\n<td><span style=\"font-family: arial\">50 ml<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">NaCl<\/span><\/td>\n<td><span style=\"font-family: arial\">3.4<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Hepes<\/span><\/td>\n<td><span style=\"font-family: arial\">3.75<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Vitamin solution (<a title=\"Vitamin solution product page\" href=\"https:\/\/www.lifetechnologies.com\/order\/catalog\/product\/11120037?ICID=search-product\" target=\"_blank\">Life Technologies<\/a>)<br \/>\n<\/span><\/td>\n<td><span style=\"font-family: arial\">5 ml<\/span><\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\"><em><span style=\"font-family: arial\">Bring to 400 ml and pH to 7.2 with NaOH<\/span><\/em><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">NaHCO<sub>3<\/sub><\/span><\/td>\n<td><span style=\"font-family: arial\">1.1<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">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>)<br \/>\n<\/span><\/td>\n<td><span style=\"font-family: arial\">5 ml<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Glutamic acid<\/span><\/td>\n<td><span style=\"font-family: arial\">0.12<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">L-Glutamine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.67<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Glycerol<\/span><\/td>\n<td><span style=\"font-family: arial\">0.38 ml<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Proline<\/span><\/td>\n<td><span style=\"font-family: arial\">0.32<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Hemin<\/span><\/td>\n<td><span style=\"font-family: arial\">1.5 ml<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">2-\u03b2-mercaptoethanol<\/span><\/td>\n<td><span style=\"font-family: arial\">7 \u03bcl<\/span><\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\"><em><span style=\"font-family: arial\">Bring to 425 ml, sterile filter and add 75 ml heat-inactivated FBS (<a title=\"FBS product page\" href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/f7524?lang=en&amp;region=GB\" target=\"_blank\">Sigma<\/a><em>)<\/em><br \/>\n<\/span><\/em><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<ul>\n<li>Grow bloodstream form <em>T. brucei<\/em> to ~2&#215;10<sup>6<\/sup> cells per ml in a 10 ml culture volume<\/li>\n<li>Pellet the culture at 1000 g for 10 minutes and resuspend in 5 ml DTM\/CCA, giving a final cell density of 4&#215;10<sup>6<\/sup> per ml<\/li>\n<li>Place the culture in a sealed flask at 27\u00b0C<\/li>\n<li>Over the first 24 hours the cell population remains static, subsequently the cells will typically double approximately every 20 hours (MITat 1.2, clone 221a); therefore, cultures are normally diltuted daily 1:1 in DTM\/CCA<\/li>\n<li>Differentiated cells are normally stable for 10 to 14 days, after which the culture collapses irrevocably<\/li>\n<\/ul>\n<p><em>NB: Unlike bloodstream form <\/em>T. brucei<em>, procyclics are sensitive to growth at low density, and differentiated cells need to be maintained at higher than 1&#215;10<sup>6<\/sup> cells per ml &#8211; they can be grown to at least 1&#215;10<sup>7<\/sup> cells per ml.<\/em><\/p>\n<p>&nbsp;<\/p>\n<table width=\"100%\" border=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td><span style=\"font-family: arial\"><strong> 10xDTM<\/strong><\/span><\/td>\n<td><span style=\"font-family: arial\">grams per litre<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">KCl<\/span><\/td>\n<td><span style=\"font-family: arial\">4<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">CaCl<sub>2<\/sub><\/span><\/td>\n<td><span style=\"font-family: arial\">2<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">NaH<sub>2<\/sub>PO<sub>4<\/sub>.H<sub>2<\/sub>O<\/span><\/td>\n<td><span style=\"font-family: arial\">1.4<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">MgSO<sub>4<\/sub>.7H<sub>2<\/sub>O<\/span><\/td>\n<td><span style=\"font-family: arial\">2<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Sodium pyruvate<\/span><\/td>\n<td><span style=\"font-family: arial\">1.1<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Phenol red<\/span><\/td>\n<td><span style=\"font-family: arial\">0.1<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Hypoxanthine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.14<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Alanine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.09<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Arginine.HCl<\/span><\/td>\n<td><span style=\"font-family: arial\">1.26<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Asparagine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.15<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Aspartic acid<\/span><\/td>\n<td><span style=\"font-family: arial\">0.14<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Cysteine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.24<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Glutamic acid<\/span><\/td>\n<td><span style=\"font-family: arial\">0.15<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">L-Glutamine<\/span><\/td>\n<td><span style=\"font-family: arial\">3<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Glycine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.08<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Histidine.HCl.H<sub>2<\/sub>O<\/span><\/td>\n<td><span style=\"font-family: arial\">0.42<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Isoleucine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.52<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Leucine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.52<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">LycinexHCl<\/span><\/td>\n<td><span style=\"font-family: arial\">0.73<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Methionine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.15<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Phenylalanine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.32<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Proline<\/span><\/td>\n<td><span style=\"font-family: arial\">0.06<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Serine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.11<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Threonine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.48<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Tryptophan<\/span><\/td>\n<td><span style=\"font-family: arial\">0.1<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Tyrosine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.36<\/span><\/td>\n<\/tr>\n<tr>\n<td><span style=\"font-family: arial\">Valine<\/span><\/td>\n<td><span style=\"font-family: arial\">0.46<\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n","protected":false},"excerpt":{"rendered":"<p>Adapted from Overath et al\u00a0 (1986) European Journal of Biochemistry 160:175 Materials 10xDTM (store in&#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-905","page","type-page","status-publish","hentry","odd"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.9 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>BSF to procyclic differentiation - Alsford Lab<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" 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