Transfection/ Transduction of IPSc

Transduction Protocol (using lentivirus or retrovirus)

Materials:

Lentiviral or retroviral vector
Polybrene (hexadimethrine bromide)
iPSCs
Culture medium (specific to iPSCs)
Plate (e.g., 6-well or 12-well)

Procedure:

Day 0: PreparationPlate iPSCs in a culture plate the day before transduction to achieve 60-70% confluence on the day of transduction.

Day 1: TransductionThaw the viral particles on ice if stored frozen. Add polybrene to the culture medium (final concentration usually around 4-8 µg/mL) to enhance viral entry. Replace the iPSC culture medium with the medium containing the viral particles and polybrene. Incubate the cells with the virus at 37°C in a humidified atmosphere with 5% CO₂ for 6-12 hours.

Day 2: Post-transductionReplace the transduction medium with fresh iPSC culture medium. Continue to culture the cells, changing the medium daily until the cells are ready for downstream applications or selection.

Transfection Protocol (using lipofection)

Materials:

Lipofection reagent (e.g., Lipofectamine 3000, FuGENE)
Plasmid DNA or RNA
iPSCs
Opti-MEM or other serum-free medium
Culture medium (specific to iPSCs)
Plate (e.g., 6-well or 12-well)

Procedure:

Day 0: PreparationPlate iPSCs in a culture plate the day before transfection to achieve 60-70% confluence on the day of transfection.

Day 1: Transfection Prepare the DNA/RNA-lipid complex:Dilute the plasmid DNA or RNA in Opti-MEM or other serum-free medium. Dilute the lipofection reagent in Opti-MEM or other serum-free medium. Combine the diluted DNA/RNA with the diluted lipofection reagent and incubate for 15-20 minutes at room temperature to allow complex formation. Add the DNA/RNA-lipid complex to the iPSCs dropwise. Incubate the cells with the transfection complex at 37°C in a humidified atmosphere with 5% CO₂ for 6-8 hours.

Day 2: Post-transfectionReplace the transfection medium with fresh iPSC culture medium. Continue to culture the cells, changing the medium daily until the cells are ready for downstream applications or selection.

Charlie Amoia

Yes, you typically dissociate induced pluripotent stem cells (iPSCs) into single cells using Accutase before plating them for transduction or transfection. Here's a general procedure you can follow:

Preparation: Pre-warm the Accutase and culture medium to room temperature or 37°C.

Aspirate Medium: Remove the old culture medium from your iPSC culture.

Add Accutase: Add enough Accutase to cover the cell layer and incubate at 37°C. The duration can vary, but typically around 5-10 minutes.

Monitor Dissociation: Gently tap the dish or flask to help dislodge the cells and monitor under a microscope to ensure cells are dissociating into single cells.

Neutralize Accutase: Add an equal volume of culture medium to neutralize the Accutase.

Centrifuge: Collect the cells and centrifuge at a low speed (e.g., 300 x g for 5 minutes) to pellet the cells.

Resuspend Cells: Carefully aspirate the supernatant and resuspend the cell pellet in fresh culture medium or transfection/transduction medium.

Count and Plate: Count the cells to determine viability and density. Plate the cells at the desired density for your transduction or transfection experiment.

Transduction/Transfection: Proceed with your transduction or transfection protocol.

Dissociating the cells into a single-cell suspension is crucial for achieving uniform transduction or transfection efficiency.

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