Protocol 1014 GEMINI™ X2 & ECM® 830 HT ELECTROPORATION PROTOCOL Cell Line: Primary Rat Hippocampal Neurons Application or Transfectant: DNA, plasmid, GFP-expressing ______Cell Preparation: Dissociated neurons were prepared from rat hippocampal slices (BrainBits). Rat hippocampal slices were incubated for 30 min in 2.5% trypsin and 100 µl 30 mg/ml DNase and then rinsed 4–5 times with Hibernate-E, no Ca2+ media plus B27 (BrainBits). Cells were triturated using large and small bore flame polished glass pipettes, chunks were allowed to settle, then the supernatant was transferred to new tubes and cells counted. Preparations commonly yield 95% neurons.

For electroporation, resuspend 100,000 neurons 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. Add 90–100 µl cells to each well of an electroporation plate, and add 1–5 µg plasmid to each well for a total volume of no more than 120 µL. ______Square Wave Electroporation Settings: Voltage Mode: Low Voltage (LV) Set Voltage: 1st pulse: 140 V, 2nd pulse: 340V Set Pulse Length: 6 ms Set Number of Pulses: 1 Pulse Field Strength: 700 V/cm, 1700 V/cm ______Electroporation Procedure: Electrode gap: 2 mm gap 96-well Electroporation Plate, Item # 45-0450 Total sample Volume: 90–100 µl cells, total volume <120 µl per well after addition of plasmid DNA Amount of transfectant: 1–5 µg plasmid DNA per well Temperature: Room Temperature Gemini Pulse: Tap plate handler icon. Using numeric keypad icon, type the number of columns being pulsed, and tap the Green Check icon to save. Program the HT plate handler with the number of pulses (for pre-pulse resistance check and DC pulses) displayed in the green message box. Tap Go icon to run protocol. ECM 830 Pulse: To pulse an entire plate set parameters listed above and then press the automatic Start to activate charge and pulse (HT200). On the plate handler (HT100) press the column select curser to the next column, the light will flash on that column and press start to activate pulse charge. Continue this until the entire plate is completed or up to the last column containing samples. Post Treatment: Immediately transfer cells to a 96-well plate containing neurobasal complete medium (NBC) supplemented with 1:50 B27), 2 mM Glutamax™ medium, 25 mM KCl, in Neurobasal-A

medium (Gibco, Thermo Fisher Scientific) preincubated at 37°C and 5% CO2.

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Results: It was possible to successfully electroporate very small numbers of neurons, only 25,000 per transfection well. Transfection efficiencies averaging 26.8% with greater than 30% viability were also reliable across an entire 96-well plate. ______Reference: Buchser, W. J., Pardinas, J. R., Shi, Y., Bixby, J. L., & Lemmon, V. P. (2006). 96-Well electroporation method for transfection of mammalian central neurons. BioTechniques, 41(5), 619-624.

Compiled by: M. Ng, BTX Technical Support, 4/18/2017