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Marina E. Wolf, Ph.D.
Professor and Chair
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PREPARATION

A. Prepare solutions

1) 100 ml PBS 1X
90 ml H2O sterile
10 ml PBS 10X
Store at 4°C

2) 50 ml CMF Buffer
45 ml Sterile H2O
5 ml PBS 10X
250 ml 20% Glucose
500 ml Fungizone
250 ml Gentamicin
Filter and store at 4°C

3) 100 ml 10 % FBS Glia Media
45ml MEM (Sigma)
5ml FBS (Gibco)
845ul 20% Glucose (sterile)
500ul Glutamine (200mM stock, Gibco)
250ul Gentamicin (Gibco)
20ul Insulin (sigma) final conc 0.01 mg/ml (stock concentration 25mg/ml of sterile water)
Filter

4) Papain Enzyme Solution (Total volume 2.5 ml)
1.87 ml Cysteine Water
52 ml Papain Suspension - before adding, sonicate for 15" and vortex for 5 min)
500 ml H&B (5X)
2.5 ml 0.5M Kynurenate
5 ml 3M HCl
Filter with 0.2 mM filter. The pH should be ~6.8. Keep at RT for 30'- 60'.

5) H&B Solution (5X) 50ml

Filter and store at 4°C

6) Cysteine Water

B. Prepare Other Supplies

1) Coat flasks with poly-d-lysine
Coat each flask with poly-d-lysine (0.1 mg/ml; solution shown below)
Incubate at 37°C overnight
Remove Poly-d-Lysine
Rinse 2x with sterile H2O and air-dry the flasks for 2hrs, store at 4°C until use.

Poly-d-Lysine solution (0.1 mg/ml)

5 mg Poly-d-Lysine
50ml ddH2O
store at -20°C

2) Prepare Wax-plate
Re-melt and sterilize the wax-plate (Petri dish with wax) under UV light (2hrs or overnight)

DISSECTION, DIGESTION, TRITURATION AND PLATING

1) Postnatal (P2-3) rats are anesthetized by hypothermia on ice and brains are removed into ice-cold phosphate-buffered saline (PBS) and bubbled with O2 continuously. The forebrain is split sagitally at the midline. The meninges are removed and the cortex is isolated. The cortex is cut into 1mm3 cubes with a scalpel blade, transferred to ice-cold CMF solution.

2) After dissection take tissue under the hood and transfer tissue (with long glass pipette, sterile) into 15 ml sterile tube.

3) Rinse tissue 4X with cold CMF, swirl gently to rinse. (Change pipette for each rinse). Take off CMF slowly.



4) Remove as much CMF as possible. Add 1 ml -1.5ml of the papain enzyme solution.

5) Incubate at 37°C for 45 min. Shake gently by hand intermittently.

6) Filter 5 ml of FBS

7) Prepare Inactivation Solution

3.5 ml Hanks BSS

1.0 ml Filtered FBS

500 ml DNase

Filter

8) Add approximately 2 ml CMF to tube with tissue and remove

9) Rinse 2 more times with CMF, and remove CMF

10)Add 1.5 ml of Inactivation Solution

11)Triturate tissue with a 9 inch Pasteur pipette followed by a 22G needle attached to a 3cc syringe

12)Using the syringe, gently layer cell suspension on top of the filtered FBS (~4 ml of FBS in the 15 ml tube).

13)Spin at 1,000 RPM for 10 min at 4°C

14)Discard as much supernatant as possible and suspend the pellet in 2ml of 10% FBS Glia Media

15)Check and count cells under microscope, do viability test

16)Cells are plated in poly-d-lysine coated flasks at 400,000 cells/cm2 final concentration in 10% FBS Glia Media (2 pups produce about 5 flasks).

17)Incubate at 37°C for 1.5h

18)Rinse the flask with ice cold 10% FBS Glia Media (about 5ml/flask)

19)Coat the flask with fresh 10% FBS Glia Media (about 7ml/flask)

Media is replaced every 5-7 days, when the confluence of the cells is about 50% of the flask surface. After the first feeding in 10% FBS Glia Media (step 17), feed the glia culture with 4% FBS Glia Media

SIGMA CATALOGUE NUMBERS

Cysteine C-1276 MW 157.6
Polysine-D-lysine P-7280 MW 30,000-70,000
Insulin I-5500 MW 5733
Hank's Balanced salt solution H-8389
Fetal Bovin Serum (FBS) F4135

GIBCO CATALOGUE NUMBERS
MEM 11090-081
Gentamicin 15710-064 (10mg/ml; stock as purchased)
L-Glutamine 25030-081 (200mM; stock as purchased)
Fungizone 15290-018 (250ug/ml; stock as purchased)

WORTHINGTON BIOCHEMICAL CORP
PAP Papain suspension LS003126 (52.1mg/ml, 22.1u/mg)
DNAse I LS002006 (3.017u/mg)