{"id":720,"date":"2013-09-06T12:48:23","date_gmt":"2013-09-06T11:48:23","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=720"},"modified":"2021-11-15T12:36:47","modified_gmt":"2021-11-15T11:36:47","slug":"ec50-analysis","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/ec50-analysis\/","title":{"rendered":"EC50 analysis"},"content":{"rendered":"

Adapted from Raz et al<\/i> (1997) Acta Trop<\/i> 68<\/b>:139<\/a>; see also, Baker et al<\/i> (2011) Mol Biochem Parasitol<\/i> 176<\/b>:55<\/a><\/p>\n

What you should end up with…\"EC50<\/a><\/i>Protocol<\/i><\/b><\/p>\n

    \n
  1. Place 200 \u00b5l media into each well in column 1, and 100 \u00b5l into all the wells in columns 2-11<\/li>\n
  2. Dilute the selective agent to twice the required high concentration and place 200 \u00b5l into each of the wells in column 12<\/li>\n
  3. Take 100 \u00b5l from each well in column 12, transfer to column 11 and pipette up and down three times to mix; repeat, generating a two-fold descending dilution series from column 12 to 3, inclusive<\/li>\n
  4. Dilute\u00a0 your wild-type and test T. brucei<\/i> cell lines to 4×103<\/sup> cells per ml<\/li>\n
  5. Place 100 \u00b5l cells into each well in columns 2-12 (final concentration of 2×103<\/sup> cells per ml)<\/li>\n
  6. Place at 37\u00b0C in a 5% CO2<\/sub> incubator for ~66 hours<\/li>\n<\/ol>\n

    After 66 hours add 20 \u00b5l 0.125 mg\/ml resazurin<\/a> (Sigma) to each well and incubate for a further 6 hours; the resultant colour change in the presence of living cells (from blue to pink) can be quantified on a plate reader: excitation, 530 nm; emission, 585 nm; filter cut-off, 570 nm<\/p>\n

    NB: after the final 6 hour incubation, plates can be placed at -20C and analysed the next day, if necessary
    \n<\/i><\/p>\n

    Analysis<\/i><\/b><\/p>\n

    Process your data in Excel (see analysis template<\/a>);\u00a0the output from the reader needs to be converted into \u2018% inhibition\u2019 and reorganised for\u00a0GraphPad Prism.<\/p>\n

    Using the ‘Analysis’ tab in Graphpad Prism…<\/p>\n