BrainBits Kits Primary Neuron Protocol

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BrainBits^® Kit Primary Neuron Culture Protocol

Please find enclosed a sterile: 

• 2 ml tube containing one pair of E18 Sprague Dawley rat hippocampus* or cortex in 2 ml of Hibernate^® EB (Hibernate E^®/B27^®/GlutaMAX™;   BrainBits^® HEB 500ml).  Use tissue immediately for highest cell yield, however, tissue can be stored for one week at 4-8^oC.
• 15 ml tube containing 12 ml of NbActiv1™ (Neurobasal^®/B27^®/GlutaMAX™) culture medium (BrainBits^® NbActiv1 500ml).
• protective sleeve containing one sterile 9” silanized pasteur pipette with a fire polished tip (BrainBits^® FPP).
• 2 ml latex bulb
• 5 ml vial of Hibernate^® E without Calcium (BrainBits^® HE-Ca 500ml).
• 6 mg vial of Papain (BrainBits^® PAP).
• pair of poly-D-lysine coated coverslips (BrainBits^® PDL) in contact lens cases (PDL side up). Additional substrate is prepared in a sterile environment
• by coating with 50 µg/ml poly-D-lysine (0.15 ml/cm²) (Sigma P6407) and incubating at room temperature for at least 1 hour (up to 20). Aspirate the PDL, rinse once with ddH₂O, aspirate the ddH₂O and air dry.

*NbActiv1™ medium for hippocampal tissue also contains 25 µM glutamate. Subsequent media exchanges should omit glutamate. 

Not supplied but required: 

• Laminar Flow Hood; 5% CO₂ Incubator; 20X Phase Microscope; 30^oC Water Bath (Shaker Optional); Centrifuge (200 x G); Trypan Blue (Sigma T8154); Hemacytometer (Sigma Z359629); 15 ml Sterile Centrifuge Tube; 0.5 ml Centrifuge Tube; Ice

Preparations (Room Temperature in a Sterile Hood)

1. Prepare cell dissociation solution by adding 3 ml of HE-Ca directly into the 6 mg vial of Papain (2 mg/ml). Recap the vial, gently mix, and place in a 30ºC water bath for 10 min to dissolve. Remove from the water bath and place on ice to cool for 10 min.
2. Add 80 µl of Trypan Blue to a 0.5 ml centrifuge tube for Step 9.

Cell Dispersal (Room Temperature in a Sterile Hood)

1. With the pasteur pipette, remove the tissue with minimal HEB media and place in the cell dissociation solution. Return excess HEB media to vial.
2. Seal the cell dissociation solution vial and incubate in a 30^oC water bath for 10 minutes. Gently swirl every 5 min.
3. With the pasteur pipette, remove the tissue with minimal cell dissociation solution and place the tissue back into the vial containing HEB media.
4. With the pasteur pipette, draw the tissue with the HEB medium into the pipette and immediately dispense contents into the same vial taking care to avoid air bubbles. Repeat for ~1 min (90% tissue dispersal).
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 media 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 37^oC, 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, 37^oC, CO₂ equilibrated NbActiv1™ every 3-4 days.

Viability Assay (Optional):

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