Functional Genomics & RNAi

Determining Relative Transduction of Adherent Cells Using MISSION® TurboGFP™ Transduction Particles

Product No. SHC003V

Protocol provided by MISSION® Team

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Introduction

MISSION® TurboGFP™ control particles enable fast determination of the appropriate transduction conditions for efficient transduction of cells with lentiviral particles. Note: The CMV promoter is known to function poorly in stem cells, lymphocytes, and primary neurons. For these cell lines observed GFP signal significantly underestimates the transduced population. Thus optimal transduction conditions can still be determined, but transfection efficiency will be underestimated. For these cell lines utilize "Titering by Transfer of Puromycin Resistance (58 kb pdf)." 

Materials

  • TurboGFP control particles (SHC003V)
  • Polybrene (H9268)
  • Cells (in log growth and at 50% confluence on the day of transfection)
  • Eppendorf tubes for serial dilutions of virus
  • Media
  • Tissue culture incubator—37 °C, 5% CO2, 100% relative humidity
  • Fluorescence Microscope with GFP filters

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Protocol

Day 0
  0.1   Count cells and plate in a tissue culture plate such that 24 hours later cells will be approximately 50% confluent.
 
Day 1
  1.1   Thaw the lentiviral particles at room temperature.
  1.2   Mix by gently tapping the tube several times with finger.
  1.3   Store the lentiviral stock on ice.
  1.4   Prepare ten-fold serial dilutions of the TurboGFP™ viral preparation and add to the designated wells.
  1.5   Add fresh media containing polybrene at a final concentration of 8 µg/ml to the cells.
  1.6   Incubate 18–20 hours at 37 °C in a humidified incubator in an atmosphere of 5% CO2.
 
Day 2
  2.1   Remove the viral-containing media from wells and add fresh media.
  2.2   Incubate cells for an additional 24–48 hours to allow for GFP expression. Ensure that adequate time has been allowed for protein expression. Robust expression of TurboGFP™ is typically observed 72 hours post-transduction.
 
Day 3–5
  Each day observe cells under fluorescent microscope. The well with the highest percentage of cells expressing GFP is recommended MOI for transduction.  

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Tips

Day 0

  • Include a non-transduced negative control well, where no virus is added.

Day 3

  • Make sure to allow sufficient time for the expression of TurboGFP™.

Figure

LNCaP seven days post-transduction image
Figure 1: LNCaP 7 days post-transduction with TurboGFP at MOI of 1.

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