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ASTROCYTES

Primary Rat Brain Astroglial Cell Culture from Hibernate Tissue

 

(According to Greg Brewer, Southern Illinois Univ. Sch. Med., hiber1.pro)

Please find enclosed one 2 ml tube containing embryonic day 18 Sprague/Dawley or Fischer 344 rat hippocampus or cortex in 2 ml B27/Hibernate. Tissue can be stored at 4-8^oC for one week. Also find 12 ml Neurobasal/10%  horse serum/3mM Glutamax. Prepare substrate by coating with poly-D-lysine (0.15 ml/cm2, 50 ug/ml water, 135 kD, 1-20 hr. e.g. Sigma P) and rinse one time with sterile 18 Mohm deionized water and let dry.

To prepare Astrocytes:

 

1. From the tube with the brain tissue, remove 1 ml medium and save for step 3, being careful not to remove the tissue.
2. With 1 ml pipettor with sterile blue plastic tip, or silanized 9 inch pasteur pipet with tip barely fire polished (preferable), suck the tissue with medium into the pipet and immediately dispense the contents back into the same container. Take care not to create bubbles. Repeat this trituration step about 10 times or until most all pieces of tissue are dispersed. Higher survival can be obtained by incubating the tissue for 30 min. at 30oC in 2 mg/ml papain (Worthington) in Hibernate, without B27, followed by trituration in Hibernate/B27.
3. Add back 1 ml medium that you removed in step 1 and mix.
4. Let undispersed pieces settle by gravity for 1 min.
5. Transfer supernatant to a new sterile 15 ml tube.
6. Spin 1100 rpm (200 x G), 1 min. Discard supernatant.
7. Flick the tube to disperse the pellet of cells. Resuspend pellet in 1 ml Nuerobasal /10% serum /3 mM Glutamax (12 mL provided; more available from www.Invitrogen.com, 1-800-955-6288).
8. Aliquot 20 ul and mix with 20 ul 0.4% trypan blue.
9. Count in hemacytometer:     =           cells/ml.
10. Dilute cells with Neurobasal/ serum/Glutamax  to plate 15 x 10³ cells/2 cm² of substrate in 0.4ml/2 cm² substrate (or whatever density you desire. Plating at higher densities will result in a mixture of neurons and astrocytes) 
11. Incubate 37^oC, 5% CO₂, 9% oxygen (20% oxygen is O.K.)
12. After 4-6 days astrocytes will be nearly confluent and ready to harvest or pass.  If desired, you can change 50% of the medium to Neurobasal / 2% Horse Serum/0.5mM Glutamax in preparation to harvest the next day for transfer to neuron cultures.  If expansion is desired, harvest cells with 0.05% trypsin in Hibernate minus calcium, 37^o, 5 minutes.  Pellet cells as in step 6 and continue with steps 7-11,

Viability assay: viability=green cells per unit area/(total cells plated per unit area) or survival (green cells/(green + red cells)):

1. Rinse 2 times with PBS or HBSS/1 g/l glucose/1 mM pyruvate/1 mM NaHCO₃/10 mM Hepes, 0.4 ml/2 cm² of substrate
2. From an acetone stock of 15 mg/ml fluorescein diacetate (Sigma), add 15 ul (1:100 dilution of the stock) into 1.5 ml HBSS. From an aqueous stock of 4.6 mg/ml propidium iodide, add 15ul of the stock into the same 1.5 ml HBSS (1:100 dilution). Add 44uL of that dilution to each well with 0.4 ml HBSS (further 1:10 dilution).
3. After about 1 min., count using Nikon B1A filter or other blue excitation appropriate for fluorescein fluorescence. Green cells are live neurons. Small red nuclear stain indicates a dead cell.
4. If desired, fix and stain with 0.25% Coomassie blue R in ethanol/HAc/ water (45/10/45), 1 min., rinse with 10% HAc, aspirate and dry.

Please report any questions or problems to:

Dr. Gregory J. Brewer

voice: 217-789-9313
fax: 217-789-9314
brainbits@brainbits.biz

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