Transfection --- suspension cells

Edited by Chang Zhu

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).