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In this video, we demonstrate how to achieve efficient and gentle intracellular delivery using the CellPore™ Transfection System.

The CellPore™ Transfection System utilizes compressed air to flow cells through microchannels embedded within the cartridge. This temporarily disrupts the cell membrane and facilitates the delivery of target materials directly into the cytosol. Refer to the Product Information Sheet that comes with your CellPore™ Transfection Kit for recommended pressure parameters for your cell type.

Here is the list of required materials and equipment: A CellPore™ Transfection System with an appropriate nitrogen or zero-air gas cylinder; a CellPore™ Transfection Kit, such as the CellPore™ Transfection Kit 300 containing CellPore™ Delivery Medium, CellPore™ Fluorescein Isothiocyanate Dextran or FITC-Dextran, and a pack of 12 CellPore™ Delivery Cartridges (each cartridge comes pre-assembled in its collection tube); the appropriate cell culture medium; appropriate labware including pipettes, a mini-centrifuge, plasticware and cultureware.

Part 1: On the day of transfection.

The CellPore™ Transfection System requires an adequate supply of compressed air in order to operate. Before proceeding, ensure the threads of the cylinder are clean and free from any debris or visible damage. Insert a new cylinder via the gas access port located on the left side of the instrument. A bit of wiggle can help fully insert the cylinder. Once fully inserted, turn the cylinder clockwise to fully engage the valve. Continue turning until the cylinder is unable to rotate any further. Refer to CellPore.com for details on ordering compatible gas cylinders. For continuous operation, it is recommended to keep a spare nitrogen or zero-air compressed gas cylinder on hand.

On the day of transfection, wipe down the CellPore™ instrument with 70% ethanol before placing it in a working biosafety cabinet. Install the safety shield that covers the nozzle. Proper installation of this safety device is required for operation. Power on the CellPore™ Transfection System and wait for the system to initialize. Before operating the instrument, confirm that the gas supply is sufficient. The indicator should display at least one bar in order to operate normally.

Select a user profile from the control bar. The list of experiments associated with the selected profile is displayed. Create a new experiment by tapping the “Add” function. In the new experiment window, define each sample by inputting the pressure and time parameters. Refer to the Product Information Sheet for recommendations on finding the optimal pressure for your cell type.

In this example experiment, we will run a five-point pressure sweep for transfecting unactivated T cells using the recommended three seconds of applied pressure per sample.

Part 2: Preparing unactivated T cells.

Standardized cell isolation methods are important considerations for obtaining best results when using the CellPore™ Transfection System. When working with fresh or cryopreserved cells, it is recommended to rest the cells in an appropriate culture medium for a minimum of one hour in the incubator. After resting, perform a cell count.

Transfer 14 million T cells into a new tube. This quantity represents the exact number required for five reactions plus two controls. Spin down the cells in a centrifuge. Carefully remove the supernatant, taking care not to disturb or aspirate the cell pellet. Serological pipettes can first be used to remove the majority of the medium. However, it is recommended to use a small-volume pipettor such as a P-200 or P-1000 to collect the residual medium. Vacuum aspirators are not recommended at this stage as this can lead to cell loss. Resuspend the cell pellet in 350 μL CellPore™ Delivery Medium. Confirm the input cell number by performing a cell count. Adjust if necessary. Set aside a 50 μL aliquot of T cells as an untreated control. This represents 2 million cells. Refer to the Product Information Sheet for more detailed information, including a list of recommended EasySep™ Isolation Kits for ready-to-use cells and additional notes and tips to consider when preparing your cells.

Part 3: Preparing the cell-cargo mixture.

Transfer 15 μL of CellPore™ FITC-Dextran to the remaining 300 μL cell suspension. Mix well. The final concentration of FITC-Dextran will be 0.1 mg/mL. Other molecules such as proteins, mRNA, or gene editing complexes can be added at this stage depending on the application. Ensure the total volume of cargo added does not exceed 10% of the total volume. In this case, 30 μL.

Part 4: Running the samples.

Transfer 50 μL of the prepared cell and cargo mixture to a new delivery cartridge. Insert the pipette tip all the way to the bottom and dispense. Close the cap of the cartridge insert and insert it into the arm of the CellPore™ instrument. Run the sample by tapping the run button for the first sample programmed for 30 psi. The instrument will apply the set pressure to the sample in the cartridge.

Once complete, retrieve the cartridge from the instrument. The sample will be in the collection tube. Run the cartridge in a mini-centrifuge to maximize sample recovery. Alternatively, gently tap the tube to collect the sample at the bottom of the collection tube. Remove and dispose of the used cartridge insert from the collection tube. Add 150 μL of CellPore™ Delivery Medium. Close the cap on the collection tube. Repeat this process, loading the next cartridge and running the remaining samples under 50, 70, 90, and 110 psi conditions. Ensure a new cartridge and collection tube are used for each sample.

Once complete, the remaining 50 μL cell and cargo mixture is used as an endocytosis delivery control. Transfer 150 μL of CellPore™ Delivery Medium to this sample as well as to the untreated aliquot set aside earlier. Transfer the tubes to the incubator for at least 30 minutes. Some cell types may benefit from a longer rest period, up to two hours at this stage. Refer to application protocols for more specific cell- handling recommendations.

Part 5: Preparing samples for downstream analysis.

Depending on the cell type and cargo, transfection results can be immediately analyzed after the resting period. It is recommended that a viability dye that has minimal spectral overlap with FITC be included to facilitate analysis of FITC-Dextran cargo delivery efficiency and cell viability. If cells are required to be cultured for downstream analysis, exchange the cells into an appropriate cell culture medium and incubate until ready for analysis.

Explore how CellPore™ can transform your cellular engineering research with its advanced intracellular delivery technology. Discover more at CellPore.com.