Primary Neuron-Glia Co-Culture

From LPDwiki

About this protocol: This protocol outlines the steps and materials involved in culturing a primary neuron-glia mixed culture taken from Day 0-1 Sprague Dawley Rats. This includes sample preparation (specifically pertaining to tissue culture plastic, glass, planar gold, and nanoporous gold), cell plating, and cell attachment. Please see the Perinatal Rat Pup Dissection protocol and Primary Neuron-Glia Co-Culture Media Preparation protocol for information on harvesting and making the media for these cultures.

Necessary Materials

1. 0.5 mg/mL Poly-L-Lysine

2. Plating Media

3. Growth Media

4. Autoclaved (Sterile) MilliQ Water

5. Sterile Tissue Culture Plates

  • Purchased from many sources such as: Corning and Falcon.

6. Plasma Cleaner

  • Plasma cleaner used in these experiments is a Harrick Plasma PDC32-G.

7. Deionized Water Source

Procedure

Important: THIS PROTOCOL MUST BE STARTED THE DAY BEFORE CELLS WILL BE COLLECTED The Biosafety Cabinet (BSC) must be used for the preparation, plating, and maintenance of the primary neuron-glia co-culture. Please follow the Biosafety Cabinet Start-up procedure before beginning this protocol.

Sample Preparation

This sample preparation applies to all materials used. Special instructions are italicized.

0. Nanoporous gold samples should be dealloyed following the np-Au Dealloying protocol, and subsequently kept in deionized water for 7 days to allow nitric acid to escape the film.

  • Deionized water should be replaced with fresh deionized water every 24 hours

1. Submerge sample in 70% EtOH solution for 5 minutes.

  • DO NOT submerge planar gold and nanoporous gold samples in 70% EtOH solutions. Start at step 3.

2. Remove sample and dry completely under nitrogen flow.

3. Plasma clean samples for 1 minute on each side with one extra minute on the top side of the sample.

4. Move samples into tissue culture plates.

5. Label with: (Do not write over the wells with samples in them)

  • Date plated:
  • Culture Name: Primary Neuron-Glia Co-Culture
  • Your name:
  • Expected day of fixation:

7. Do not forgot your controls!

Poly-L-Lysine (PLL) and Plating Media Incubation (PM)

1. Add 500 uL of 0.5 mg/mL PLL solution to each sample (including any tissue culture plastic you are culturing on!).

2. Place the samples with PLL solution into an incubator at 37°C and 5%CO2 for 4 hours.

3. Remove the samples from the incubator and place back into the BSC.

4. Aspirate PLL solution from each sample and replace with 500 uL of autoclaved milliQ water for 1 minute.

5. Aspirate and repeat 6 times.

6. Aspirate the water from each sample and replace with 500 uL of Plating Media (PM).

7. Place the samples with PM into an incubator at 37°C and 5%CO2 overnight until cell plating.

Cell Plating

1. Gather cells following the Perinatal Rat Pup Dissection protocol.

2. Dilute cells from the stock solution to an adequate density for plating.

  • All experiments performed by Chris Chapman have used a cell density of 522 cells per square millimeter.
Cells per well:
24 well plate 12 well plate 6 well plate
100,000 cells 200,000 cells 500,000 cells

3. Aspirate the PM from the samples and replace with 500 uL of the cell dilution.

4. Spread cells over the surface of your wells by sliding back and forth and side to side.

  • DO NOT slide in a circular motion. This will cause the cells to aggregate towards the middle of the well.

5. Place samples into an incubator at 37°C and 5%CO2 for 4 hours to allow for attachment.

6. Remove the samples from the incubator and place back into BSC.

7. Aspirate PM from the samples.

  • This step needs to be done both carefully and quickly!

8. Replace with 500 uL of Growth Media (GM).

  • This step needs to be done both carefully and quickly!

9. Place samples into an incubator at 37°C and 5% CO2 until the desired fixation time is reached.

Culture Maintenance

At Day in vitro 3

1. Remove the samples from the incubator and place into BSC.

2. Replenish GM by adding 500 uL of fresh media to the wells.

At Day in vitro 7

1. Remove the samples from the incubator and place into BSC.

2. Aspirate approximately 500 uL of growth media from each of the wells.

  • This step needs to be done both carefully and quickly!

3. Replenish GM by adding 500 uL of fresh media to the wells.

Repeat this every 3 and 4 days respectively.

Cleaning Up

1. Dispose of used conical tubes and pipettes into the biological waste bin.

2. Bleach and thoroughly rinse out the used aspirator and hang up to dry.

3. If you are done with work in the BSC, follow the Biosafety Cabinet Shutdown procedure.