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Optimization of Antibiotic Selection Using a Cytotoxicity Profile

互联网2013-11-13

590

实验试剂

1. Cell Culture Media

2. Antibiotics puromycin (P9620) or G418 (A1720)

实验设备

1. Tissue culture incubator—37 °C, 5% CO₂ , 100% relative humidity

实验材料

1. Cells - in log growth and at 50% confluence on the day of transfection

实验步骤

1. Day 0
1)	Seed cells at 50% confluence per well in complete media. Seed cells in 3 rows x 11 columns for cytotoxicity assay.
2)	Return cells to incubator and incubate overnight.
2. Day 1
1)	Pre-warm 5 ml of full media.
2)	Make three independent dilutions in complete media of puromycin yielding 11 concentrations of puromycin: 0, 1, 2, 3, 4, 5, 6 7, 8, 9, 10 µg/ml. Each dilution should have at least 110 µl as the final volume.
3)	Remove cells to incubator
4)	Remove media from cells
5)	Replace media with antibiotic containing media
6)	Return steps 1.1 through 1.6
3. Day 3
1)	Repeat steps 1.1 through 1.6.
4. Day 5
1)	Repeat steps 1.1 through 1.6.
5. Day 7
1)	Repeat steps 1.1 through 1.6.
6. Day 10
1)	Determine viability in each well by either cell counting or viability assay.
2)	The minimum concentration of puromycin resulting in complete cell death after 7-10 days of selection with puromycin should be used for that cell type for transduction with shRNA viral particles.

注意事项

If cells started to round but did not detach from the plate surface after addition of puromycin:

1. Rounding could be a sign of cells dying without detaching from the surface yet. These cells should detach with more time.

2. If cells require higher concentration of puromycin then the recommended 1-10 μg/ml, check the expiration date on the puromycin and avoid multiple freeze-thaw cycles (<5).

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