Biomod/2013/OSU/protocol/cell analysis
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<a href="http://openwetware.org/wiki/Biomod/2013"> << Back to Team Pages</a>
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Cell Analysis Procedure
Loading Efficiency
- Setup a 2 mM Daunorubicin Solution (Given Concentration of Solution)
- Add Origami shapes at a concentration of 5.0 nM
- Incubate for 40 hours
- Centrifuge at 20.0 xG for 50 minutes @ 20 deg C
- Isolate Orange-red precipitate from solution - PEG (Keep solution—gives concentration of daunorubicin not absorbed by origami/used in loading efficiency calculation)
- Read Absorption at 488 nm with a microplate reader (FRET Setting)
Cellular Methods
- Redissolve origami shapes in PBS to form Stock solution (~1 mM Daunorubicin and~1.25 nM origami)
- Dilute with cell culture medium to form final Daunorubicin concentrations of 5 to 100 µm
Cellular Viability (MTT)
- Seed 96-Well plate with CLL cells and incubate overnight
- Add Origami Shapes, double stranded DNA, or free doxorubicin at specified concentration to cells for 12 hours
- Wash with PBS
- Incubate for 24, 36, 48 hours
- 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)
- Read absorbance at 450 nm using microplate reader
Dead Cell Staining (TIRF)
- Seed 8-Well plate with CLL cells and incubate overnight
- Add Origami shapes, double stranded DNA, or free doxorubicin at specified concentration to cells for 12 hours
- Wash with PBS
- Incubate for 48 hours
- Incubate with SYTOX green (10 µM) for 20 minutes at 37ºC
- Image using Fluorescence Microscopy
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