Optimal Cell Density Determination (cells/ml) for Transduction in Osteosarcoma Cell KHOS Using MISSION® LentiExpress™ Optimization Plate
(Product No. SHXC01)
shRNA

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Protocol courtesy of Harvard Medical School, Department of Orthopedic Surgery (Dr. Zhenfeng Duan MD, PhD)



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Materials

  • 0.25% Trypsin EDTA solution
  • LentiExpress Optimization Plate (Product No. SHXC01)
  • 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
  • Microtubes

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Protocol

Day 0

0.1 Thaw LentiExpress optimization plate at room temperature while completing steps 0.2 through 0.7.

0.2 Fill and label four 1.5 ml tubes with 300 µl of complete media.

0.3 Trypsinize KHOS cells and dilute to 160,000 cells/ml in 600 µl of complete media.

0.4 Serially dilute by two fold KHOS cells into the labeled tubes.

0.5 Add polybrene to a final concentration of 11.3 µg/ml to each tube (including the stock tube of KHOS cells).

0.6 Mix cells and polybrene.

0.8 Repeat step 0.7 in column 3 using the next highest concentration of cells, and continue until all concentration of cells have been plated onto the LentiExpress optimization plate.

0.9 Cover and transfer the optimization plate to the tissue culture incubator. Incubate overnight.


Day 1

1.1 Remove optimization plate from the incubator.

1.2 Gently remove the media and properly dispose of the media.

1.3 Replace media with 100 µl of complete media per well that contains cells.

1.4 Cover and return optimization plate to the tissue culture incubator.


Day 2

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.

Day 4

4.1 Repeat 2.1 through 2.5.


Day 6

6.1 Observe cells using fluorescence microscopy.


MISSION is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.
LentiExpress is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.



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