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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 4-8oC.
- 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; 30oC 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)
- 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.
- Add 80 µl of Trypan Blue to a 0.5 ml centrifuge tube for Step 9.
Cell Dispersal (Room Temperature in a Sterile Hood)
- With the pasteur pipette, remove the tissue with minimal HEB media and place in the cell dissociation solution. Return excess HEB media to vial.
- Seal the cell dissociation solution vial and incubate in a 30oC water bath for 10 minutes. Gently swirl every 5 min.
- With the pasteur pipette, remove the tissue with minimal cell dissociation solution and place the tissue back into the vial containing HEB media.
- 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).
- Let undispersed pieces settle for 1 min.
- Transfer supernatant containing dispersed cells to a sterile 15 ml tube. Leave ~50 µl of HEB containing debris.
- Spin 1100 rpm (200 x G), 1 min. Discard supernatant leaving ~50 µl of HEB media 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.
Viability Assay (Optional):
- 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).