Non-Adherent Cell Lines Protocol

Preparing the CytoView-Z Plate - Antibody Coating

1. Pre-coat (100 µl per well is recommended) the entire well surface of the CytoView-Z plate using the relevant antibody for your cell type to anchor the non-adherent cells to the impedance plate surface. E.g. anti-CD40 for Daudi cells (4 µg/mL) or anti-CD71 for K562 cells (5 µg/mL).  Tip: Reconstitute the relevant antibody in PBS for the cell line that is cultured in accordance to supplier recommendations.

2. Incubate the antibody-coated plate at 4°C, overnight.

3. Aspirate the antibody solution from the plate.

4. Add 100 µl of complete medium to the plate, and add 8 mL of sterile water to the on-plate reservoirs to increase humidity.

5. Dock the plate in the Maestro Z to measure the media only (MO) Baseline. Transfer the plate to a biosafety cabinet when the Baseline is complete.

Culturing Non-Adherent Cell Lines for Transfer to CytoView-Z Plate

6. Thaw and culture the cells of interest in accordance with supplier recommendations, passaging as needed.

7. Take the flasks of cultured non-adherent cells from the 37°C incubator, and mix the suspension gently, but thoroughly with a serological pipettor.

8. Remove a sample of the cell suspension and count the cells using a hemocytometer to determine both the viability and total number of viable cells.  Tip: Ensure the cells are evenly suspended before removing an aliquot to count.

9. Transfer the cell suspension to a 15 ml conical tube.

10. Centrifuge the cell suspension at 250 x g for 5 minutes.

11. Aspirate the supernatant, being careful not to disturb the cell pellet.

12. Dilute the cell suspension in complete medium to a working concentration of cells per 100 µl. The working concentration should be the number of cells per well necessary to achieve 100% confluence within 24 hours, however this may vary based on specific experimental goals. Note: It is recommended to run a cell density sweep with a cell type first to determine the optimal number of cells for the working concentration and to inform future experiments.

Plating Non-Adherent Cells onto the CytoView-Z Plate

13. Undock the CytoView-Z plate from the Maestro Z once the MO baseline has been collected, and transfer the plate to a biosafety cabinet.

14. Transfer the cell suspension to a trough for easy access by a multichannel pipette. Alternatively, divide the cell suspension evenly into microcentrifuge tubes that can be used with a multichannel pipette, providing enough volume to seed the cells with a 100 µl addition per well.  Tip: Be sure to mix the cell suspension thoroughly before any addition to ensure even distribution of the cells. Dispense the cells directly in the middle of the well.

15. After seeding cells on the plate, leave it to rest in the biosafety cabinet for 1 hour at room temperature. Tip: High well-number microtiter plates are sensitive to thermal gradients, which can cause edge effects.

16. Dock the plate and impedance measurements will begin automatically upon plate engagement.

17. For optimal cell health, 50% of the media should be changed after 48 hours.

Required materials

Consumables

Equipment