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BrainBits® Frozen Neuron Culture Protocol for 4 Million Cell Vials
Enclosed is one vial of E18 Sprague Dawley Rat cortex or hippocampus stored in 500 µl of cryopreservative. Upon arrival, store on dry ice or liquid Nitrogen until ready for use.
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 at room temperature for at least 1 hour (up to 20). Aspirate the poly-D-lysine, rinse once with ddH2O, aspirate and air dry.
- Aliquot 30 µl of Trypan Blue (Sigma: T8154) into a 0.5 ml tube for Step 4 in Cell Plating.
- Allow NbActiv1™ medium (Neurobasal®/B27®/GlutaMAX™) to come to room temperature (BrainBits® NbActiv1 500ml).
- Only thaw one vial of cells at a time (leave remaining vials stored on dry ice or liquid Nitrogen).
- Place one vial of cells in a 37ºC water bath until pellet thaws (~ 3 min).
- Remove immediately being careful not to shake or vortex the vial
Cell Plating (Room Temperature in a Sterile Hood)
- Transfer 0.85 ml of thawed cells to a 50 ml sterile centrifuge tube making sure not to create bubbles.
- Add 7.65 ml of room temperature NbActiv1™ (dropwise over 2 min) to the 50 ml sterile centrifuge tube from Step 1.
- Mix by gently swirling the 50 ml centrifuge tube.
- Aliquot 30 µl of cell solution into the 0.5 ml tube containing 30 µl of Trypan Blue (1:2 dilution).
- Immediately plate 0.2 ml/cm2 substrate (range 0.15-0.5 ml/cm2).
- Incubate cells and NbActiv1™ (0.2 ml/cm2 of substrate) at 37oC, 5% CO2, 9% O2, 95% humidity (or ambient O2)
- Count cells (Step 5) using a hemacytometer (calculate cells/ml).
- After a 45-180 min incubation remove cells and NbActiv1 from the incubator.
- Aspirate ~90% of original NbActiv1™ from the substrate and immediately add 0.2 ml/cm2 of equilibrated NbActiv1™.
- 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)
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Neurobasal®, B27®, and GlutaMAX™ are trademarks of Life Technologies.