Biomod/2013/OSU/protocol/cell analysis: Difference between revisions

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<h1>Cell Analysis Procedure</h1>

<h2>Loading Efficiency</h2> <ol> <li>Setup a 2 mM Daunorubicin Solution (Given Concentration of Solution)</li> <li>Add Origami shapes at a concentration of 5.0 nM</li> <li>Incubate for 40 hours</li> <li>Centrifuge at 20.0 xG for 50 minutes @ 20 deg C</li> <li>Isolate Orange-red precipitate from solution - PEG (Keep solution—gives concentration of daunorubicin not absorbed by origami/used in loading efficiency calculation)</li> <li>Read Absorption at 488 nm with a microplate reader (FRET Setting)</li> </ol>


<h2>Cellular Methods</h2> <ol> <li>Redissolve origami shapes in PBS to form Stock solution (~1 mM Daunorubicin and~1.25 nM origami)</li> <li>Dilute with cell culture medium to form final Daunorubicin concentrations of 5 to 100 µm</li> </ol>

<h3>Cellular Viability</h3> <ol> <li>Seed 96-Well plate with CLL cells and incubate overnight</li> <li>Add Origami Shapes, double stranded DNA, or free doxorubicin at specified concentration to cells for 12 hours</li> <li>Wash with PBS</li> <li>Incubate for 24, 36, 48 hours</li> <li>Incubate with fresh serum-free medium containing 0.5 mg/mL WST-1 for 1 h at 37 °C for the cytotoxicity assay (Specific to cell-counting kit used) </li> <li>Read absorbance at 450 nm using microplate reader</li> </ol>

<h3>Dead Cell Staining</h3> <ol> <li>Seed 96-Well plate with CLL cells and incubate overnight</li> <li>Add Origami shapes, double stranded DNA, or free doxorubicin at specified concentration to cells for 12 hours</li> <li>Wash with PBS</li> <li>Incubate for 48 hours</li> <li>Incubate with SYTOX green (10 µM) for 20 minutes at 37ºC</li> <li>Image using Fluorescence Microscopy</li> </ol>


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