shRNA

Optimization of Antibiotic Selection Using a Cytotoxicity Profile

 

Protocol provided by the MISSION® team

Introduction

The appropriate concentration of antibiotic for selecting stable cell lines is different for each cell type. If the concentration for the desired cell type is unknown, a titration experiment must be performed to determine the lowest concentration of antibiotic needed to efficiently select transduced cells. In this protocol, we highlight the use of puromycin, the antibiotic utilized with the standard pLKO.1 vector. Typically, 1-10 µg/ml are sufficient to kill most untransduced mammalian cell types.  Click Here to see a recommended starting concentration for a wide variety of cell lines. This general protocol can also be utilized for determining the optimal concentration of G418 required for the custom shRNA vectors. See the custom shRNA page for more information. Higher concentrations than absolutely required of antibiotic could lead to off target effects and fewer cells for downstream analysis.

Materials

  • Cells - in log growth and at 50% confluence on the day of transfection
  • Cell Culture Media
  • Tissue culture incubator—37 °C, 5% CO2, 100% relative humidity
  • Antibiotics puromycin (P9620) or G418 (A1720)

Protocol

Day 0
0.1 Seed cells at 50% confluence per well in complete media. Seed cells in 3 rows x 11 columns for cytotoxicity assay.
0.2 Return cells to incubator and incubate overnight.
Day 1
1.1 Pre-warm 5 ml of full media.
1.2 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.
1.3 Remove cells to incubator
1.4 Remove media from cells
1.5 Replace media with antibiotic containing media
1.6 Return steps 1.1 through 1.6
Day 3
3.1 Repeat steps 1.1 through 1.6.
Day 5
5.1 Repeat steps 1.1 through 1.6.
Day 7
7.1 Repeat steps 1.1 through 1.6.
Day 10
10.1 Determine viability in each well by either cell counting or viability assay.
10.2 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.

Tips

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

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