Difference between revisions of "Primary Neuron-Glia Co-Culture Media Preparation"

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(Plating Media Preparation)
 
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7. Add 5 mL of Glutamax to Bottle A.
 
7. Add 5 mL of Glutamax to Bottle A.
  
8. Add Agitate the culture media to mix (via pipetting).
+
8. Agitate the culture media to mix (via pipetting).
  
 
9. Place the vacuum filter on the second autoclaved bottle (Bottle B) and turn the vacuum on.
 
9. Place the vacuum filter on the second autoclaved bottle (Bottle B) and turn the vacuum on.

Latest revision as of 15:56, 7 June 2016

About this protocol: This protocol outlines the steps and materials involved in creating sterile culture medium used in maintaining cultures of disassociated primary rat neocorticies. This includes B Buffer, 0.5 mg/mL Poly-L-Lysine solution, Plating Media (PM), and Growth Media (GM). Please see the Perinatal Rat Pup Dissection protocol and Primary Neuron-Glia Co-Culture protocol for information on harvesting and maintaining these cultures.

Necessary Materials

1. Neurobasal A

  • Purchased from Invitrogen (10888-022) and should be stored in 4°C upon arrival.

2. B27 Supplement

  • Purchased from Invitrogen (17504-044) and should be stored in -20°C upon arrival.

3. Heat-inactivated Horse Serum (HS)

  • Purchased from Invitrogen (26050-088).
  • Upon arrival HS should be aliquoted to 50 mL and stored at -20°C

4. Glutamax

  • Purchased from Invitrogen (35050-061) and should be stored in 4°C upon arrival.

5. 1M HEPES pH 7.5 Tissue Culture Grade

  • Purchased from Invitrogen (15630080) and should be stored in 4°C upon arrival.

6. Poly-L-Lysine

  • Purchased from Sigma (P1399) and should be stored in -20°C upon arrival.
  • Poly-D-Lysine may also be used.

7. Borax

  • Purchased from Sigma (B3545) and can be stored at room temperature.

8. Boric Acid

  • Purchased from Sigma (B7660) and can be stored at room temperature.

9. Autoclaved Glass Bottles (6x)

  • Autoclaving is done by UC Davis Veterinary Medicine Central Services located in Haring Hall.

10. 0.2 um Vacuum Bottle Top Filter (2x)

  • Bottle top filters can be purchased from UC Davis Veterinary Medicine Central Services located in Haring Hall or Sigma Aldrich (CLS430049).

Procedure

Important: The Biosafety Cabinet (BSC) must be used for the preparation of primary neuron-glia co-culture media. Please follow the Biosafety Cabinet Start-up procedure before beginning this protocol.

B Buffer Preparation

Prepare B Buffer in a tissue culture safe autoclaved bottle

1. Add 3.1 grams boric acid.

2. Add 4.75 grams borax.

3. Add 1 liter of tissue culture grade water.

4. Adjust pH to 8.5.

5. Sterile filter into a second tissue culture safe autoclaved bottle.

6. Label with: (write on tape not on the glass)

  • Date made:
  • Solution Name: B Buffer (Borax, Boric Acid)
  • Your name:

7. Store at room temperature.

Poly-L-Lysine (PLL) Solution Preparation

Outside BSC

1. Add 250 mg of PLL into a 50 mL conical tube.

2. Add 35 mL of B Buffer.

3. Shake at room temperature for ~10 minutes until PLL is dissolved.

Inside BSC

4. Place an autoclaved bottle into the BSC.

5. Attach a filter unit and add ~100 mL of B Buffer.

6. Add the dissolved PLL and fill the filter to 500 mL.

7. Sterile filer the 500 mL solution.

8. Label with: (write on tape not on the glass)

  • Date made:
  • Solution Name: 0.5 mg/mL PLL in B Buffer
  • Your name:

9. Store at 4°C. Replace after 2 months of storage.

Plating Media Preparation

1. Thaw one aliquot of both HS and B27 supplement overnight in 4°C before starting this protocol.

Inside the BSC

2. Place both autoclaved bottles and sterile filtering unit into the BSC.

3. Add 500 mL of Neurobasal A solution into one autoclaved bottle (Bottle A).

4. Add 50 mL of HS to Bottle A.

5. Add 10 mL of B27 supplement to Bottle A.

6. Add 10 mL of 1M HEPES pH 7.5 (tissue culture grade) to bottle A.

7. Add 5 mL of Glutamax to Bottle A.

8. Agitate the culture media to mix (via pipetting).

9. Place the vacuum filter on the second autoclaved bottle (Bottle B) and turn the vacuum on.

10. Slowly pour the media from Bottle A through the filter into Bottle B.

11. Turn the vacuum off and detach the vacuum filter before discarding in the biological waste bin.

12. Cap both Bottle A and B tightly.

13. Aliquot from Bottle B into 50 mL conical tubes.

14. Label conical tubes with

  • Date made:
  • Solution Name: Plating Media (PM)
  • Your name:

Outside the BSC

14. Place the aliquots into -20°C storage.

13. Place the used bottle (A) into the sink for cleaning.

Growth Media Preparation

1. Thaw one aliquot of B27 supplement overnight in 4°C before starting this protocol.

Inside the BSC

2. Place both autoclaved bottles and sterile filtering unit into the BSC.

3. Add 500 mL of Neurobasal A solution into one autoclaved bottle (Bottle A).

4. Add 10 mL of B27 supplement to Bottle A.

5. Add 5 mL of Glutamax to Bottle A.

6. Add Agitate the culture media to mix (via pipetting).

7. Place the vacuum filter on the second autoclaved bottle (Bottle B) and turn the vacuum on.

8. Slowly pour the media from Bottle A through the filter into Bottle B.

9. Turn the vacuum off and detach the vacuum filter before discarding in the biological waste bin.

10. Cap both Bottle A and B tightly.

11. Aliquot from Bottle B into 50 mL conical tubes.

12. Label conical tubes with

  • Date made:
  • Solution Name: Growth Media (GM)
  • Your name:

Outside the BSC

14. Place the aliquots into -20°C storage.

13. Place the used bottle (A) into the sink for cleaning.

Cleaning Up

1. Ensure the remaining Neurobasal A, Glutamax, and 1M HEPES is tightly capped, marked sterile, and place into 4°C for storage.

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

3. Thoroughly rinse out the used bottle and hang up to dry.

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