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Primary Neuronal Cell Culture

 

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BrainBits® Primary Neuron Culture 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 tissue immediately for highest cell yield however, tissue can be stored for one week 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):

  1. 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.
  2. Prepare cell dissociation solution by dissolving 6 mg sterile papain (BrainBits® PAP 6 mg) in 3 ml of Hibernate E-Ca (HE-Ca) without B27 (BrainBits® HE-Ca 5 ml) for a final working concentration of 2 mg/ml papain. Incubate for 10 minutes at 30ºC to dissolve.
  3. Fire polish the tip of a sterile 9” silanized pasteur pipette to an opening of ~0.5 mm (BrainBits® FPP).
  4. Aliquot 80 µl of Trypan Blue (Sigma: T8154) into a 0.5 mL tube for Step 9.

Cell Dispersal (Room Temperature in a Sterile Hood):

  1. With the silanized pasteur pipette, carefully transfer HEB solution to a sterile tube (save for Step 3) leaving the tissue with minimal HEB.
  2. Add 2 ml of cell dissociation solution to the tissue and incubate for 10 min at 30oC. Gently swirl every 5 min.
  3. Remove cell dissociation solution leaving the tissue at the bottom. Return the HEB medium from step 1.
  4. With the silanized pasteur pipette, triturate tissue for about 1 min (90% tissue dispersal) avoiding air bubbles.
  5. Let undispersed pieces settle for 1 min.
  6. Transfer supernatant containing dispersed cells to a sterile 15 ml tube. Leave ~50 µl of HEB containing debris.
  7. Spin 1100 rpm (200 x G), 1 min. Discard supernatant leaving ~50 µl of HEB containing the pellet.
  8. Disperse the pellet of cells (flick the bottom of the tube with a finger) and resuspend pellet in 1 ml NbActiv1™
  9. Aliquot 20 µl of cell solution into the 0.5 ml tube containing 80 µl of Trypan Blue (1:5 dilution).
  10. Count cells using a hemacytometer (calculate cells/ml).

Cell Plating (Room Temperature in a Sterile Hood):

  1. Dilute cells with NbActiv1™ (0.2 ml/cm2) and plate at 16,000cells/cm2 or desired concentration.
  2. Incubate 37oC, 5% CO2, 9% O2, 95% humidity (or ambient O2).
  3. After 4 days, neurons display axons and dendrites; synapses and action potentials begin at 7 days.
  4. Change ½ of the medium with fresh, 37oC, CO2 equilibrated NbActiv1™ every 3-4 days.

Viability Assay:

  1. Rinse twice with 37ºC HBSS (0.2 ml/cm2 of substrate).
  2. 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).
  3. Add 20 µl of dye mix from step 2 to every 0.2 ml of HBSS added in step 1 (1:10 dilution).
  4. 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).
  5. 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).

 



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