The following protocol has been developed for high-content screening in 96-well plates.
Prepare mammalian cells so that they are growing exponentially and are no more than 70-80% confluent before transduction.
Prepare a stock solution of hexadimethrine bromide at 2 mg/mL in water.
Add 1.6 x104 cells in fresh medium to the number of wells needed for each construct in a 96-well plate. Duplicate or triplicate wells for each lentiviral construct and control to be used.
Incubate 18-20 hours at 37 °C in a humidified incubator in an atmosphere of 5-7% CO2.
Note: The growth rates of cells vary greatly. Adjust the number of cells plated to accommodate a confluency of 70% upon transduction. Also account for the length of time the cells will be growing before downstream analysis when determining the plating density.
Remove medium from wells. To each well add 110 µL medium and hexadimethrine bromide to a final concentration of 8 µg/mL. Gently swirl the plate to mix.
Note: Hexadimethrine bromide enhances transduction of most cell types. Some cells, like primary neurons, are sensitive to hexadimethrine bromide. Do not add hexadimethrine bromide to these types of cells. If working with a cell type for the first time, a hexadimethrine control only well should be used to determine cell sensitivity.
Add 2-15 µL of lentiviral particles to appropriate wells. Gently swirl the plate to mix. Incubate 18-20 hours at 37 °C in a humidified incubator in an atmosphere of 5-7% CO2. Cells may be incubated for as little as 4 hours before changing the medium containing lentiviral particles. Overnight incubation may be avoided when toxicity of the lentiviral particles are a concern.
Note: When transducing a lentiviral construct into a cell line for the first time, a range of volume or MOI should be tested. 2, 5, 10, and 15 µL of lentiviral particles per 1.6 x 104 cells or MOIs of 1, 2, and 5 should be used to determine the optimal transduction efficiency and knockdown for each cell line (see Appendix). Transduction efficiency can be optimized using the MISSION TurboGFP Control Transduction Particles before assaying with shRNA constructs.
Remove the medium containing lentiviral particles from wells. Add fresh medium to a volume of 120 µL to each well.
Note: For cell types that do not strongly adhere to the plate, 100 µL of medium may be removed and replaced with 100 µL fresh medium.
Replace medium every 3-4 days until cells are to be assayed.
Cells may be selected and each clone may be expanded to assay for expression of shRNA.
A variety of phenotypic, enzymatic, or gene expression assays may be performed. The desired assay should be optimized prior to the high-content screen with both negative and positive controls.
Note: Due to the random integration of the lentivirus into the host genome, varying levels of TurboGFP expression or shRNA expression may be seen with different colonies. Testing a number of colonies will allow the optimal degree of expression to be determined.
Multiplicity of Infection (MOI): Multiplicity of Infection is the number of transducing lentiviral particles per cell. It is highly recommended that for each new cell type to be transduced, a range of MOI be tested. This will determine the optimal amount of lentiviral supernatant needed for efficient transduction of each cell line used.
(Total number of cells per well) x (Desired MOI) = Total transducing units needed (TU)
(Total TU needed) / (TU/mL reported on C of A) = Total mL of lentiviral particles to add to each well