Calcium Phosphate Transfection of Eukaryotic Cells   

Abstract

Materials and Reagents

1. HEK-293 cells (ATCC, catalog number: CRL-1573 ™)
   
2. Eagle's minimum essential medium (ATCC, catalog number: 30-2003 ™)
   
3. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
   
4. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C5670 )
   
5. HEPES (Sigma-Aldrich, catalog number: H4034 )
   
6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
   
7. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S5136 )
   
8. pEGFP-Actin plasmid (this is an example plasmid; Clontech, catalog number: PT3265-5 )
   Note: The pEGFP-Actin Vector expresses the EGFP-Actin fusion protein in mammalian cells; this protein is incorporated into growing actin filaments and allows for visualization of actin-containing subcellular structures in living and fixed cells. http://www.clontech.com/images/pt/dis_vectors/PT3265-5.pdf
   
9. 2x HBS buffer (see Recipes)
   
10. Solution-A (see Recipes)
    
11. Solution-B (see Recipes)
    

Equipment

1. Tissue culture plates (e.g., 35 mm polystyrene)
   
2. Cell culture incubator: 37 ºC and 5% CO₂
   

Procedure

1. Prepare 2 M CaCl₂ solution in water, filter sterilize and keep at room temperature.
   
2. Prepare the 2x HBS buffer (see Recipes).
   
3. Carry HEK-293 cells in Eagle's Minimum Essential Medium with 10% FBS.
   
4. 24 h before transfection, trypsinize and reseed log-phase cells into 35 mm tissue culture dishes. For seeding density, cells should reach 60-70% confluence for transfection.
   Note: Best confluence for different cell lines differ. For HEK-293 cells, 60-70% confluence is suggested.
   
5. 3 h before transfection, replenish cells with fresh medium.
   
6. For each DNA transfection, prepare mixtures in two separate tubes (Solution-A and Solution-B, see Recipes)
   
7. Add Solution-B slowly (drop-wise) into Solution-A while mixing Solution-A.
   Note: This is the most important step for forming DNA/calcium phosphate co-precipitate. Mix gently but thoroughly to allow formation of precipitates evenly.
   
8. After mixing the two solutions, incubate at room temperature for 20-30 min (a shorter incubation time may be used for different cell types; please determined empirically). The solution will become opaque while precipitates being formed.
   
9. Gently tap the mixture. Add the mixture directly to cells by dripping slowly and evenly into medium (a good way is to let tip touch medium surface).
   
10. Gently tilt the plate back-and-forth a couple of times to allow even distribution of added precipitate on the cell surface.
    
11. Incubate the cells at 37 ºC with 5% CO₂ for 24 h and then replenish medium.
    

Notes

1. GFP expression, if used as your positive, can be detected usually after 24-48 h of cell growth.
   
2. Transfection efficiency (when using HEK293 cells and the above mentioned sample plasmid) can reach up to 90-100%.
   

Recipes

1. 2x HBS buffer

   50 mM	HEPES
   280 mM	NaCl
   1.5 mM	Na2HPO4

   Adjust pH to 7.0 using HCl. Filter sterilize. The solution can be freezed/thawed once for future use.
   
2. Solution-A
   100 μl 2x HBS
   
3. Solution-B
   1-5 μg DNA (e.g., pEGFP-Actin plasmid as suggested above)
   12.2 μl of 2 M CaCl₂
   ddH₂O to bring volume up to 100 μl, pipet gently to mix
   Note: Titration of DNA should be carried out to obtain the best efficiency of transfection.
   

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Graham and van der Eb (1973) and Jordan et al. (1996). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee  [see Chen et al. (2009)].

References

1. Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
   
2. Graham, F. L. and van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456-67.
   
3. Jordan, M., Schallhorn, A. and Wurm, F. M. (1996). Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4): 596-601.