The rat pheochromocytoma PC12 cell line is widely applied as a model system to study a variety of neuronal functions. Upon addition of nerve growth factor (NGF), these cells undergo differentiation characterized by an increase in acetylcholinesterase (AChE) activity, and extension of neurite-like processes. Transfection methods are often used to deliver genes into these cells in order to study their interactions. In doing so it is important that the cells are not altered by the transfection and maintain their ability to differentiate. In this report we show that the use of Lipofectamine™ LTX to transfect PC12 cells does not affect neurite outgrowth or AChE activity post transfection.

The rat pheochromocytoma PC12 cell line is widely applied as a model system to study a variety of neuronal functions. Upon addition of nerve growth factor (NGF), these cells undergo differentiation characterized by an increase in acetylcholinesterase (AChE) activity, and extension of neurite-like processes. Transfection methods are often used to deliver genes into these cells in order to study their interactions. In doing so it is important that the cells are not altered by the transfection and maintain their ability to differentiate. In this report we show that the use of Lipofectamine™ LTX to transfect PC12 cells does not affect neurite outgrowth or AChE activity post transfection.

PC12 Cells (ATCC, Manassas, VA, USA) maintained in Neurobasal™ Media (Invitrogen Cat# 21103-049) supplemented with 1%

L-Glutamine (Invitrogen Cat# 25030-081), 10% Fetal Bovine Serum (FBS) (Invitrogen Cat# 16000-044) and 5% Donor Horse Serum (Sigma, St. Louis, MO, USA).

Lipofectamine™ LTX Reagent (Invitrogen Cat# 15338-100 or 15338-500)

PLUS™ Reagent (Invitrogen Cat# 11514-015)

N2-Supplement (Invitrogen Cat# 17502-048)

Poly-D-lysine (BD, Franklin Lakes, NJ, USA)

Nerve Growth Factor 7S (NGF 7S),

Murine, Natural (Invitrogen Cat# 13290-010)

Vivid Colors™ pcDNA™ 6.2/EmGFP Plasmid DNA (Invitrogen, Carlsbad, CA, USA)

Opti-MEM® I Reduced Serum Medium (Invitrogen Cat# 31985-062)

Amplex® Red Acetylcholine/Acetlycholinesterase Assay Kit (Invitrogen Cat# A12217)

Phosphat-Buffered Saline (PBS) (Invitrogen)

Cell Lysis Buffer (Invitrogen)

PC12 cells were maintained in Neurobasal Media supplemented with 1% L-Glutamine, 10% FBS, and 5% Donor Horse Serum. Differentiated cells were maintained in the Neurobasal Media with 1× N2-Supplement. Cells were passaged without trypsin by careful tituration to break up clusters and wash cells. Cells were passaged 1:5 twice per week to maintain moderate confluence. On the day of transfection, 20,000 cells per well were plated in 96-well plates.

Forty-eight hours after transfection cells were transferred to 24-well plates coated with poly-D-lysine for cell adherence. Twenty four hours later, 100 ng/ml Nerve Growth Factor 7S was added to the growth media. We measured changes in the total number of neurite-bearing cells, AChE levels, compared to untreated control cells 3 days following exposure to NGF.

Transfection of pcDNA6.2/EmGFP plasmid into PC12 cells was optimized by titrating the amount of Lipofectamine™ LTX Reagent. Twenty-four hours after transfection, cells were allowed to become adherent, and then they were stimulated with media containing 100 ng/ml NGF.

Transfections were performed in duplicate. For each transfection sample in 96-well format, 100 ng of pcDNA6.2/EmGFP plasmid DNA was diluted in 25 µl Opti-MEM® I Reduced Serum Medium, 0.1 µl of PLUS™ Reagent was added, and the DNA/PLUS™ Reagent solution was mixed and allowed to incubate for 5 min. Lipofectamine™ LTX Reagent was then added to the well and gently mixed by pipetting up and down, and the lipid/DNA/PLUS™ Reagent complex was allowed to form by incubating at room temperature for 30 min. Twenty microliters of the DNA-lipid complex was added to each well of the 96-well plates containing cells.

PC12 cells were collected after 3 day exposure to NGF, washed three times with PBS, and lysed in 100 µl Cell Lysis Buffer. AChE activity was measured using the Amplex® Red Acetylcholine/AChE Assay Kit. A volume of 25 µl per reaction from each lysate was used. Activity was determined using a standard curve and recorded as nmol/min/mg protein.

Figure 1. Morphology of the PC12 cells after transfection with Lipofectamine™ LTX Reagent using a fluorescent microscope to visualize GFP expression in these cells. (Click to enlarge)

After incubation for 3 days, the proportion of neurite-bearing cells was counted. Processes longer than 1.0× cell body diameters were counted as neurites. For every data point, the mean value was calculated from five random field observations of two replicate experiments and at least 50 cells were counted per field.

The treatment of PC12 cells with NGF significantly increase the proportion of neurite-bearing cells compared to control, 2% vs. 65%. Lipofectamine™ LTX Reagent had nearly equivalent numbers (59%) of neurite-bearing cells as the NGF control. Exposure to NGF caused an increase in AChE activity from 11 to 420 nmol/min/mg proteins in untransfected cells and an increase to 463 for transfected cells.

Commonly used markers for neuronal cell differentiation are increased AChE activity and increased neurite outgrowth. Here we have optimized transfection of a pcDNA6.2/EmGFP reporter into PC12 cells. Our results show equivalent NGF induced neuronal differentiation in transfected and un-transfected PC12 cells, indicating that neuronal differentiation of these PC12 cells as measured by neurite outgrowth and AChE activity are not affected by Lipofectamine™ LTX Reagent.

Other cell specific protocols can be found at www.invitrogen.com/transfection.

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