The Hinck Lab
Lab Members 2002 -2003

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Hippocampal Cell Culture

Adapted from Culturing Nerve Cells, 2nd Edition; Banker and Goslin 1998

pages 352-356

Note, we have switched from the glia feeder layer to neurobasal media with B27 supplement.

Coverslips

"Assistent" German glass coverlips (number 1001)

1. Rinse 2 x 10 min with milli-Q water.

2. Soak in concentrated nitric acid for 24 hours

3. Wash 5 times over 3 hours with sterile milli-Q water

4. Bake overnight at 225C.

5. Dot each coverslip with 3 parafin (autoclaved) drips

6. Expose to UV light for 1 hour.

7. Treat with poly-l-lysine overnight (1mg / 1ml borate buffer pH 8.5 filter sterilized)

8. Wash with sterile milli-Q water 2x over 2 hours

9. Add Neuronal Plating Medium - MEM (with Earle's salts) with 10% heat inactivated horse serum, 0.6% glucose, 1mM pyruvic acid

10. incubate at 37C + 5% CO2

 

Hippocampus

Dissect away the hippocampus from E18 rat embryos. Collect 8-10 hippocampi on ice in Hepes Buffered Salt Solution in 15ml centrifuge tube.

Dissociation and Plating of Hippocampal Neurons

Adjust HBSS volume in centrifuge tube to 5ml. Add 0.5ml 2.5% trypsin and incubate for 15min at 37C. Draw off the trypsinized HBSS and add 5ml fresh HBSS. Let stand 5 min, repeat HBSS wash two more times. Bring the final volume of HBSS to roughly 1.5ml.

Dissociating the hippocampi

Dissociate hippocampi with fire-polished Pasteur pipette (to 1/2 the initial diameter) — roughly 15-25 pipettes up and down or until no obvious chunks remain.

Determine the cell density.

Plating Cells

We typically want to plate 2x10^5 cells per small petri dish containing 5 coverslips so divide cells/ml by this desired # cells per dish to get the volume of homogenized cell solution to apply to each dish. We find it easiest to plate at least 75ul per dish.

Remove the coverslips in Plating Media from the incubator. Gently drip the proper volume of homogenized hippo cells over the coverslips. We find it easiest to plate at least 75ul per dish. Place back in the incubator (37C). 2-3 hours later, flip each of the coverslips upside down into culture dishes containing Neurobasal media plus B27 supplement and pen/strep/glutamine. Return these dishes to the incubator. We find that cells grown upside down in this manner collect less debris and are generally better looking after 2 weeks

Maintenance

We do not add cytosine arabinoside to these cultures as glial proliferation is rarely a problem. We do not feed the cell cultures or change the media. We typically begin infecting cells with sindbis virus around day 14 in vitro and proceed to immunostaining 1-2 days later.