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Effects of Passage Number on Cell Line Transfection

Linda Jacobsen1 and Peyton Hughes2*
1Roche Applied Science, Indianapolis, USA; 2ATCC, Manassas, USA

*Corresponding author

An important factor in the success of any transfection experiment is the quality of the cells. As a key experimental component, the cell line can be the factor with the highest variability, affecting the reliability and reproducibility of results.

In transfection experiments, the passage number of cell lines can affect not only transfection efficiency, but also protein expression. To eliminate these concerns, Roche Applied Science recommends using freshly obtained, authenticated cell lines from ATCC, and monitoring passage level. Cell lines are being increasingly recognized as standard experimental reagents and as essential components to ensure reproducible and reliable results in life science research [1–4]. Manufacturers of quality life science products routinely and exclusively use ATCC cell lines for product development and optimization. The performance of an optimized product can suffer when used with cell lines of inferior quality.

The data shown in Figure 1 demonstrate low- and high-passage RAW 264.7 (ATCC® TIB-71™) cells can be transfected equally well, but protein expression is significantly reduced in the high-passage cells. RAW 264.7 (ATCC® TIB-71™) cells were transfected with a plasmid encoding for luciferase expression at passage numbers 5 (low passage) and 74 (high passage) using FuGENE® HD Transfection Reagent. Three different volumes (4 µl, 6 µl, and 10 µl) of the same complex (5:2 ratio of FuGENE® HD Transfection Reagent to DNA) were added to all cells. Similar expression levels were observed 24 hours post-transfection at either passage number. However, luciferase expression dropped off significantly 48 hours post-transfection in the high-passage cells (Figure 1a). Minimal inhibition of cell proliferation was observed in low-passage cells with all three volumes of complex. In contrast, growth inhibition was observed in the high-passage cells when 6–10 µl of the complex was added. This effect on proliferation was not observed when less complex was added (Figure 1b).

For more information about passage number effects, and problems with use of unauthenticated cells, visit the ATCC Online Cell Resource at


1.   Hartung T et al. (2002) ATLA 30:407–414

2.   Chatterjee R (2007) Science 315:928–931

3.   Nardone RM (2006) Eradication of cross-contaminated cell lines: A call to action. Available at:

4.   O’Brien SJ (2001) Proc Natl Acad Sci USA 98:7656–7658

For ATCC Genuine Cell Cultures™ contact ATCC or an authorized distributor.


This article was originally published in Biochemica 3/2007, pages 32. ©Springer Medizin Verlag 2007

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