Introduction

The expression of proteins in cells using promoter driven cDNAs is a widely used approach for studying the function of proteins and analyzing molecular networks. Over the past 20 years, a number of approaches have been developed to allow transfection of cDNA containing plasmids into cells, especially cell lines. However, primary cells, such as neurons and T cells, have been resistant to most transfection methods, requiring the use of time-consuming and expensive viral-based methods. Neurons have been particularly challenging to transfect, with low efficiency of transfection up until the past few years (1). Recently, Amaxa (2) has introduced the nucleofection method, which achieves neuronal transfection efficiencies of >20% (3,4). A single cuvette is used in this method to electroporate a million or more neurons at a time. Each electroporation requires the use of an expensive proprietary reagent. For testing multiple genes, millions of neurons have to be harvested, and electroporations have to be performed one at a time. This presents two major problems for testing many genes. First, the repetitive action of electroporating single samples, and then manually placing them correctly in their destination, lends itself to variability and error. Second, the extended time it takes to sequentially transfect each gene leaves the neurons in a toxic environment leading to even lower viabilities and efficiencies.

High content screening (HCS) uses automated acquisition of images of cells in multiwell plates combined with detailed quantitative image analysis to obtain multiple parameters about cell morphology and molecular expression. HCS is ideally suited for screening various kinds of libraries for their effects on cell proliferation, apoptosis, or other cell biological processes. It is ideal for quantitatively studying the morphology of neurons and how various agents alter process growth. The paucity of efficient methods for testing cDNAs in mammalian neurons using HCS has restricted the usefulness of HCS approaches to drug and compound libraries and inhibited genome style studies. To overcome this roadblock, we have sought to develop an inexpensive method that would allow multiple transfections at once—with identical conditions except for the gene to be transfected. This electroporation approach involves a mixture of a minimal set of components, all at reasonable volumes and with small numbers of cells, such that experiments can be performed quickly with multichannel pipets or 96-well liquid handlers.

Materials and Methods

Primary Neuron Culture

Postnatal day 8–11 mouse cerebella were prepared as described previously (5). Briefly, cerebella were harvested from ketamine-euthanized mice and minced with a razor blade. The cerebellar pieces were incubated in 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) for 20 min at 37°C, with occasional swirling. The trypsin was inactivated by adding horse serum to 10% and diluted with Hank's balanced salt solution (HBSS; 5.4 mM KCl, 0.44 mM KH₂PO₄, 131 mM NaCl, 4.2 mM NaHCO₃, 0.34 mM Na₂HPO₄, 10 mM HEPES). The cells were triturated sequentially with large- and small-bore flame-polished glass pipets in the presence of 1 mg/ mL DNase I (Sigma, St. Louis, MO, USA). Hoechst dye (Invitrogen) was added during this step. The cells were spun and resuspended in HBSS for counting. Centrifugation steps were all performed at 115× g for up to 7 min. Solutions and cells were kept at room temperature throughout the procedure. Preparations yielded >90% cerebellar granule neurons (CGNs).

Rat hippocampal slices were purchased from BrainBits LLC (Springfield, IL, USA), and dissociated neurons were prepared using a modified version of their protocol. Briefly, half hippocampal slices were incubated for 30 min in 2.5% trypsin and 100 µL 30 mg/mL DNase and then rinsed four to five times with Hibernate-E, no Ca²⁺, media plus B27 (BrainBits LLC). Cells were triturated using flame-polished small- and large-bore glass pipets. After triturating, chunks were allowed to settle, and then the supernatant was transferred to new tubes, and the cells counted. Volumes were kept small (<2 mL) throughout the process. Preparations commonly yield >95% neurons.

Transfection

Typically, 100,000 neurons were resuspended in 100 µL intracellular buffer (INB) solution (135 mM KCl, 0.2 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, 5 mM EGTA, pH 7.3), which results in about 3 nM free calcium. Cells (90–100 µ L) are added to a 96-well, 2-mm gap, electroporation plate (HT-P96-2; Harvard Apparatus/BTX, Holliston, MA, USA), and 1–5 µg plasmid cDNA were also added to each well for a total volume of no more than 120 µL. The plate is sealed with a 3M ScotchPad™ tape sheet (Qiagen, Valencia, CA, USA). All solutions were removed from the cold 10 min prior to transfection to allow them to come to room temperature. The electroporation plate was placed inside the Model HT-200 plate handler attached to an ECM 830 square wave pulse generator (Harvard Apparatus/BTX). This system was used to deliver pulses to the plate, one column at a time. For CGNs, one pulse was delivered with 340–350 V, 900 µs pulse length. For hippocampal neurons, two pulses were delivered, each with 900 µs pulse lengths and an approximate 2-s interval between. The first pulse was 140 V and the second 340 V.