Making Glial-coated coverslips

5/12/06

Glial Medium:

500mL DMEM (high glucose)

50mL FBS

5mL pen/strep

5mL glutamine

Glial cells are harvested from P0 or older brains by taking cortex, dissociating with papain just as is done for cortical neuronal cultures, then plating 0.5 million or 1 million cells in 10mL glial medium on a non-coated plastic dish. Only the glia will stick under these conditions. Change the medium the next day to remove dead unstuck cells, and feed biweekly replacing half of the medium. 1 million plates will be confluent in 2-3 weeks, 0.5 in 3-4 weeks. Confluent glial plates can be split at least once, with a 1:5 split reconfluent in 1 week, 1:10 in 2 weeks, 1:20 in 3 weeks. Do not use glial plates that have lots of dead round white cells, or little black microglial “spiders” as these cells tend to be unfriendly to the neurons.

Cells need to grow at 5% CO2, so use upper incubator on the right.

Overnight before splitting glia, coat acid washed coverslips (see coverslip preparation protocol) with Poly-D-Lysine AND laminin. I never coat less than overnight and always use both substrates only once. Wash the coverslips on the plate 2X with tissue culture sterile water before plating glia.

Prewarm medium, 1X Ca-Mg free HBSS, and trypsin for at least 20’ in tc water bath.

Remove medium from cells and wash 2X with 5mL Ca-Mg free HBSS. Make sure to spray the solution on the side on the plate not right down on the cells so they don’t wash off. Minimize the time the cells are dry after removing the medium.

Remove HBSS and add 2mL trypsin. Return plate to incubator for 3-5’.

Gently shake plate to see that cells are coming up. You can also watch cells come off the substrate by looking on the microscope.

Leave trypsin on and add 10mL serum-containing medium. Spray around the plate to spray all the cells off the plate. Move to 15mL tube and spin cells down 5’ at 2K in tc centrifuge.

Aspirate medium and add 10mL fresh serum-containing medium. Resuspend cells 1X with 10mL pipette, then 1X with a P200 tip on the end of the 10mL pipette.

Use the hemacytometer to count cells. All the cells in 25 large squares x 10 to the 4^th equals the number of cells you have per mL.

Plating density depends on the day you will want to plate neurons on the coverslips. The earliest you can add neurons is the day after plating the glia. For this time frame, plate all the glia from 1 10cm dish onto 1 24 well plate of coverslips. (So resuspend the trypsinized cells from a confluent 10 cm dish in 12mL medium and put 0.5mL on each well of a 24 well plate of coverslips). You should get about 3 million glia from a confluent plate, so that is about 125,000 per well.

More commonly, we will split glia on Friday for plating neurons in the middle of the next week. In this case, plate 45,000 glia per well. Note that this means one confluent 10cm plate of glia is sufficient to coat 3 24 well plates of coverslips. Watch the coverslips to make sure the glia become confluent by the time the neurons are added.

Neurons are plated in medium that is primarily serum-free, so glial growth will slow when you add the neurons. The day after plating neurons, add AraC at 5uM to the medium, which will block further glial division.