Astrocytes Culture Protocol

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 BrainBits^® Primary Astroglial 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 NbASTRO (Neurobasal^®/Horse Serum/ GlutaMAX™). Use tissue immediately for highest cell yield however, tissue can be stored for one week at 4-8^oC.

 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 30^oC. 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 of Astroglial medium (BrainBits^® NbASTRO).
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 NbASTRO (0.2 ml/cm²) and plate at 7,500cells/cm² or desired concentration. Note: Plating at higher densities will result in a mixture of neurons and astrocytes.
2. Incubate 37^oC, 5% CO₂, 9% O₂, 95% humidity (or ambient O₂).
3. After 4-6 days, astrocytes will by 90% confluent and ready to harvest or pass.
4. Transfer to neuron cultures: 24 hours prior, change ½ of the medium to Neurobasal^®/B27^®/GlutaMAX™ (BrainBits^® NbActiv1^TM).
5. For expansion: Harvest cells with 0.05% trypsin in HE-Ca, (37^oC, 5 min). Pellet cells as in step 7 and continue with steps 8-10 in Cell Dispersal and Steps 1 & 2 in Cell Plating.

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);Brewer & Price (1996) Neuroreport 7:1509-1512 (PMID: 8856709); and Boehler et al. (2007) Neuron Glia Biol. 3:127-140 (PMID: 18345351).