Suspension cells can be transfected using protocols similar to the protocols for adherent cells. Change of medium is achieved by centrifuge suspension cells for 10 minutes at 250-1000 g. Remove supernatant and change to fresh medium. Cell density is usually maintained at 1.0-3.0 x 10⁵ cells/ml. At this density, number of cells in a 1 ml culture is roughly comparable to the number of cells on a 35 mm plate. Scale accordingly.

This protocol uses the DOTAP Liposomal Transfection Kit from Roche.

1. Dilute cells 1:2-1:5 the day before transfection. Estimate cell density.

2. For each 10⁵ cells, dilute 2.5 mg DNA to 0.1 mg/ml in HBS (HEPES-buffer saline: 20 mM HEPES, 150 mM NaCl, pH7.4).

3. In a separate sterile tube, dilute 15 ml DOTAP in HBS to a final volume of 50 ml.

4. Transfer the DNA/HBS to DOTAP/HBS and mix by pipetting.

5. Incubate at room temperature for 15 minutes.

6. Mix the DOTAP/DNA with 1-2 ml of culture medium.

7. Spin down cells and replace medium with the culture medium containing DOTAP/DNA. Alternatively add the culture medium containing DOTAP/DNA dropwise to the cells and by gentle rocking.

8. Return cells to incubator, incubate for 3-6 hours.

9. Replace medium with fresh culture medium (without DOATP/DNA).