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BrainBits® Dissociated Primary Neuronal Plating Protocol
Enclosed is one pair of E18 Sprague Dawley Rat cortex or hippocampus* (1 pair/tube) in 2 ml of Hibernate® EB (HEB; Hibernate E®/B27®/GlutaMAX™; BrainBits® HEB 500 ml) and a 12 ml tube of NbActiv1™ medium (Neurobasal®/B27®/GlutaMAX™; BrainBits® NbActiv1 500 ml). Use dissociated primary neurons immediately for highest cell yield however, cells can be stored for two weeks at 4-8oC.
*NbActiv1™ medium for hippocampal tissue also contains 25 µM glutamate. Subsequent media exchanges should omit glutamate.
Preparations (Room Temperature in a Sterile Hood):
- Prepare substrate by coating with 50 µg/ml poly-D-lysine (0.15 ml/cm2)(Sigma P6407). Incubate coated surfaces for at least 1 hour (up to 20). Aspirate the poly-D-lysine, rinse once with ddH2O, aspirate and air dry.
- Fire polish the tip of a sterile 9” silanized pasteur pipette to an opening of ~0.5 mm (BrainBits® FPP).
- Aliquot 80 µl of Trypan Blue (Sigma: T8154) into a 0.5 ml tube for Step 6.
Cell Dispersal (Room Temperature in a Sterile Hood):
- Allow 12 mL of NbActiv1™ to come to room temperature.
- Warm the 2 mL tube of dissociated primary neurons in a 30ºC water bath for 1 min.
- With the silanized pasteur pipette, transfer the suspension to a sterile 15 ml centrifuge tube and gently triturate no more than 5 times while avoiding air bubbles.
- Spin 1100 rpm (200 x G), 1 min. Discard supernatant leaving ~50 µl of HEB containing the pellet.
- Disperse the pellet of cells (flick the bottom of the tube with a finger) and resuspend pellet in 1 ml NbActiv1™
- Aliquot 20 µl of cell solution into the 0.5 ml tube containing 80 µl of Trypan Blue (1:5 dilution).
- Count cells using a hemacytometer (calculate cells/ml).
Cell Plating (Room Temperature in a Sterile Hood):
- Dilute cells with NbActiv1™ (0.2 ml/cm2) and plate at 16,000cells/cm2 or desired concentration.
- Incubate 37oC, 5% CO2, 9% O2, 95% humidity (or ambient O2).
- After 4 days, neurons display axons and dendrites; synapses and action potentials begin at 7 days.
- Change ½ of the medium with fresh, 37oC, CO2 equilibrated NbActiv1™ every 3-4 days.
- Rinse twice with 37ºC HBSS (0.2 ml/cm2 of substrate).
- Prepare dye mix from an acetone stock of 15 mg/ml fluorescein diacetate and an aqueous stock of 4.6 µg/ml propidium iodide, dilute 15 µl of each into 1.5 ml HBSS (1:100 dilution).
- Add 20 µl of dye mix from step 2 to every 0.2 ml of HBSS added in step 1 (1:10 dilution).
- After ~1 min count live cells using blue excitation appropriate for fluorescein fluorescence (green cells). Count dead cells with green excitation for propidium iodide fluorescence (small red nuclei).
- Viability = (green cells/unit area)/(total cells plated/unit area) or Survival = green cells/(green + red cells)
For additional tips please see the FAQs at www.BrainBitsLLC.com.
Neurobasal®, B27®, GlutaMAX™, and Hibernate® are trademarks of Life Technologies.
Methods based on Brewer et al. (1993) J. Neurosci. Res. 35:567-576 (PMID: 8377226)and Brewer & Price (1996) Neuroreport 7:1509-1512 (PMID: 8856709).