Protocol courtesy of Harvard Medical School, Department of Orthopedic Surgery (Dr. Zhenfeng Duan MD, PhD)
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- LentiExpress Kinase Plates (Product No. SHX001)
- 0.25% Trypsin EDTA solution
- Puromycin Solution (Product No. P9620)
- Complete Media—RPMI 1640 with 10% fetal bovine serum
- Polybrene (hexadimethrine bromide) (Product No. H9268)
- Electronic repeating pipette
- Fluorescence Microscope—capable of detecting GFP in a 96-well plate
- Tissue culture incubator—37 °C, 5% CO2, 100% relative humidity
- Tissue culture laminar flow hood—only open optimization plate in tissue culture hood
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0.1 Thaw LentiExpress kinase plate at room temperature while completing steps 0.2 through 0.7.
0.2 Trypsinize KHOS cells and dilute to 40,000 cells/ml in 8.1 ml of complete media.
0.3 Add polybrene to a final concentration of 11.3 µg/ml.
0.4 Mix cells and polybrene.
0.5 Add 70 µL to each well of a LentiExpress kinase plate.
0.6 Cover and transfer the plate to the tissue culture incubator.
0.7 Incubate overnight.
1.1 Remove the plate from the incubator.
1.2 Gently remove the media and dispose of the media in 10% bleach solution.
1.3 Replace media with 100 µL of complete media per well.
1.4 Cover and return optimization plate to the tissue culture incubator.
2.1 Make complete media with puromycin at a concentration 1 µg/ml.
Note: this was found to be optimal for this particular cell line. For other cell lines, optimal concentration of puromycin must be determined empirically. See the optimization protocol web page for an example.
2.2 Remove optimization plate from the tissue culture incubator.
2.3 Remove media from wells that contain cells.
2.4 Add 100 µL of media with puromycin to those wells.
2.5 Cover and return to tissue culture incubator for two days.
4.1 Repeat 2.1 through 2.5.
6.1 Observe cells using fluorescence microscopy.
- Stevenson, C.S. et al. Journal of Pharmacology and Experimental Therapeutics 2002, 303, 858-66.
- Martin, B.T.; Strebhardt, K. Cell Cycle 2006, 5, 2881-5.
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LentiExpress is a trademark of Sigma-Aldrich Co. LLC
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