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    Determining Relative Transduction of Adherent Cells

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    实验原理

     

    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)."

    实验试剂

     

    1. TurboGFP control particles (SHC003V)

    2. Polybrene (H9268)

    3. Media

    实验设备

     

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

    2. Fluorescence Microscope with GFP filters

    3. Eppendorf tubes for serial dilutions of virus

    实验材料

     

    Cells (in log growth and at 50% confluence on the day of transfection)

    实验步骤

     

     

    1. Day 0
     

    1)

    		  

    Count cells and plate in a tissue culture plate such that 24 hours later cells will be approximately 50% confluent.
     
    2. Day 1
     

    1)

    		  

    Thaw the lentiviral particles at room temperature.
     

    2)

    		  

    Mix by gently tapping the tube several times with finger.
     

    3)

    		  

    Store the lentiviral stock on ice.
     

    4)

    		  

    Prepare ten-fold serial dilutions of the TurboGFP™ viral preparation and add to the designated wells.

     

    5)

    		  

    Add fresh media containing polybrene at a final concentration of 8 µg/ml to the cells.

     

    6)

    		  

    Incubate 18–20 hours at 37 °C in a humidified incubator in an atmosphere of 5% CO2 .
     

    3. Day 2
     

    1)

    		  

    Remove the viral-containing media from wells and add fresh media.
     

    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.
     

    4. 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.

    注意事项

     

    1. Day 0

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

    2. Day 3

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

     

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