Plating Cortical Neurons

8/19/04

Neurons are available M, R, F (usually) from Sara. She has about 600 million cortical neurons per day on a good day. These are left on ice in the tc room with a paper towel next to the ice bucket on which you sign up for how many you want. The requests are tallied at 11:30am, at which time if the requests are less than the total available, you can plate your cells. If the requests exceed the total, you must negotiate for who gets what.

Coat plates at least 2 hours at 37degrees. PDL is made at 1mg/mL in ddH2O and stored in the vertical –80. Dilute 1mL aliquot in 50mL tc sterile ddH2O. Cover plates and put in 37 degree incubator. Remove PDL after 2+ hours, and keep to reuse up to 2 months. Wash plates 2X with ddH2O (put on and remove right away). Coated plates can be stored for up to 2 months wrapped in foil at 4 degrees.

Medium:

500mL BME (sigma)

50mL Calf serum (CS)

5mL pen/strep

5mL glutamine

6.67mL 45% glucose (tc sterile)

Make sure cap of 50mL tube is tight and invert tube of neurons several times to mix. Usual concentration of neurons is about 6 million per mL. Divide total number of neurons you need by the concentration to get mL to take. You will want 18 million neurons per plate in a total of 12mL medium. Plate 1mL/well on 12 well plate for luciferase assays. Return to incubator.

For luciferase assays transfect on DIV3-4 and stimulate for luciferase assay on DIV5-6.