Marina E. Wolf, Ph.D

Professor and Chair 
Department of Neuroscience
Chicago Medical School
Room 2.262
Building: BSB
Phone: 847.578.8659
Fax: 847.578.8515
marina.wolf@rosalindfranklin.edu
Protocols


NUCLEUS ACCUMBENS CELL CULTURE PROTOCOL

1. PREPARATION


2 Days before dissection
1.1 Prepare glass coverslips and poly-d-lysine coated plate or flasks.
Needed:
glass coverslips (Fisher brand 12mm,12-454-82)
Poly-d-lysine
1M HCl
95% EtOH
24 well plate
1) Put some clean coverslips in a small beaker with 1M HCl (~ 30ml) and sonicate for 15 min
2) Wash the coverslips with ddH2O twice
3) Wash the coverslips with EtOH 95% 2x and sonicate for 5 min
4) Rinse in fresh EtOH 95% (the coverslips can be stored at 4°C for a couple months)
5) Individually flame dry the coverslips and place in a 24-well-plate.
6) Sterilize under UV light for about 1-2 hr
7) Add 0.4 -0.5ml Poly-d-Lysine/well (24 well plate) and incubate at 37°C overnight
8) Remove Poly-d-Lysine
9) Rinse 2x with sterile H2O or PBS 1X and air-dry the plates in the hood (dark) for 2hrs, store at 4°C until use (covered with Aluminum Foil).

24 hours before dissection
1.2 Prepare Conditioned Media for NAc cells
1) Prepare 50 ml Initial Neurobasal Media
Initial Neurobasal Media is used for primary NAc cultures only on first day of culture. Fifty ml of Initial Neurobasal Media is sufficient for two plates - this amount can be obtained from 4 flasks (25cm2) of glial cells (12.5ml/flasks).
50 ml Neurobasal Media
500 ml Glutamine
250 ml Gentamicin
500 ml Fungizone
2) Filter (0.22mm filter)
3) Add 12.5 ml above media to each flask of glial cells, condition the media for 8-24 hr.
1.3 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
1.4 Autoclave the following equipment and supplies
One 16G sharp edged cannula (for making tissue punches), two 27G needles, 1 blade handle, 2 forceps, 1 spatula, 1 scissors, long and short pipettes, 500ml and 200ml tips
1.5 Wax-plate
Melt 1 wax sheet (Ted Pella, Inc. Cat # 109) on 60mm plate, sterilize the wax filled plate under UV (2h or overnight). The wax can be remelted.

The Dissection Day
1.6 Continue preparation of Conditioned Media for NAc cells (see 1.2 above).
Collect conditioned media from glial cells (to be used today; day 1 of culture) and prepare additional medium to be conditioned for use on the next day.
1) Thaw Glutamic acid and B27 under the hood
2) Make 50 ml Regular Neurobasal Media for conditioning today, use tomorrow
50 ml Neurobasal Media
500 ml Glutamine (200 mM)
250 ml Gentamicin (10mg/ml)
3) Remove Initial Neurobasal Media conditioned from glia cultures and add
500 ml Glutamic Acid
500 ml B27
4) Filter, then store at 4°C until needed.
5) Add the Regular Neurobasal Media to glial cultures to condition it for next day use.
1.7 Prepare 0.5M Kynurenic acid
Dissolve 1mg kynuerenic acid in 10.57ul 1N NaOH
1.8 Prepare Cysteine water
30 ml 0.5M CaCl2
500 ml 0.02M Cysteine
7.35 ml H2O
Filter
1.9 Prepare equipment and supplies
1) 2 ice buckets, 2 empty sterile beakers, 2 absorbent bench underpads, blade, wax plate, syringe, transfer pipette,2 beakers of PBS, paper towels
2) Put PBS at -80°C until ice crystals just start forming.
3) Get ice, set up ~15 ml CMF Buffer on ice with O2 bubbling through a 22G needle
4) Thaw 5 ml FBS and 0.5 ml DNAse
5) Set up microscope, turn on Hood, Centrifuge, water bath

2. DISSECTION, DIGESTION, TRITURATION AND PLATING
(7-8 rat pups for 2 plates)
1) Clean the wax plate with CMF and PBS 1x, put PBS with ice forming on the wax plate.
2) Put the pups in the ice bucket to anesthetize them (2-3min).
3) Take the pups out, disinfect with alcohol, cut the head off and gently remove the brain.
a) Sagittal dissection method
Put the brain in the wax plate on ice, cut it in two hemispheres along the
midline. Using anterior commissure as marker to localize the peripheral border of the NAc, punch out the tissue containing NAc out with 16G cannula, remove lateral 1/3 and medial ¼ of the cylinder, then remove remaining tissue (NAc) to the beaker containing CMF and O2.
b) Coronal dissection method
Place the whole brain, ventral surface facing up, on a wax plate (petrie dish filled with wax) on ice. It may be necessary to fix the brain in place with a needle. Make a coronal cut just rostral to where the rostral poles of the two hemispheres come together - at the level shown in Coronal Plate 5 (Atlas of Prenatal Rat Brain Development, J Altman & SA Bayer, CRC Press, 1995). Make a second coronal cut just rostral to the circle of willis - at a level that is approximately in between those shown in Coronal Plates 7 and 8. Place the resulting coronal section, caudal surface facing down, in a glass petrie dish that has been pre-chilled. Punch out the nucleus accumbens with a sharpened 16 G cannula, then transfer it to the beaker containing CMF and O2.
4) While there are 4 rats left, make up the papain enzyme solution
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 Hanks' Balanced Salt Soultion (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) After dissection take tissue under the hood and transfer tissue (with long glass pipette, sterile) into 15 ml sterile tube.
6) Rinse tissue 4X with cold CMF, swirl gently to rinse. (Change pipette for each rinse). Take off CMF slowly.
7) Remove as much CMF as possible. Add 1 ml -1.5ml of the papain enzyme solution.
8) Incubate at 36-37°C for 30 min. Mix gently by tapping with hand intermittently.
9) prepare Inactivation Solution and filter 5 ml of FBS
3.5 ml Hanks BSS
1.0 ml FBS
500 ml DNase
Filter
10) Remove papain enzyme solution using glass pipette.
11) Rinse 2x with CMF, and remove CMF.
12) Add 1.5 ml of inactivation solution
13) Triturate tissue
15x with the sterile long glass pipette
6-8x with 3cc syringe with 22G syringe needle
14) Using the syringe, gently layer cell suspension on top of the sterilized 4ml FBS in the 15 ml tube).
15) Spin at 1,000 RPM for 10 min at 4°C.
16) Get the Initial Neurobasal Media which has been stored at 4 °C and warm up under the hood.
17) After cells have spun, discard as much supernatant as possible
18) Suspend cells in 1.0 ml of Initial Neurobasal Media, using pipetman with 1000ml blue tip 4-5X gently.
19) Check and count cells under microscope, do viability test
20) Dilute to the correct concentration (4x104 cells/ml) and plate on 24 well plates (1 ml/well)
3. MEDIUM CHANGE

3.1 FIRST CHANGE
(The media is changed for the first time after 24 hr in culture.)
As described above, prepare 50 ml Regular Neurobasal Media on the day of the dissection, condition with glia cultures for 24h. After conditioning, add 500ml B27, and filter. Remove all of the old media from the wells and add 1 ml of the Regular Neurobasal Media to each well.
3.2 SUBSEQUENT CHANGES
(Following the first change, half of the media is replaced every 4-5 days.)
Prepare 25 ml Regular Neurobasal Media (for 2 flasks), condition for 24h. After conditioning, add 500 ml B27, filter. Remove 500ml of old media from each well and add 500ml fresh media to each well.
4. NOTES
1) Each medium must be conditioned for 8h-24h in Glial cultures
2) Regular NB Med must be changed every 4-5 days.
3) For cortical cell cultures, dissect cortex, cut it into 2X2X2mm cubes and follow the above protocol.
 

Solutions for Nucleus Accumbens Cell Culture
Regular NeuroBasal Medium

Ingredient

Stock Conc.

Amount

Final Conc.

NeuroBasal Media



10ml



Gentamicin

10mg/ml

50ul

0.05mg/ml

Glutamine

200mM

100ul

2mM

B27



200ul



1. Condition the medium without B27 in glial cultures (80% confluent) for 8-24 hrs.
2. Add B27, filter, store at 4 degrees C till needed.

Initial NeuroBasal Medium

Ingredient

Stock Conc.

Amount

Final Conc.

NeuroBasal Media



10ml



Gentamicin

10mg/ml

50ul

0.05mg/ml

Glutamine

200mM

100ul

2mM

B27



100ul



Glutamic Acid

2.5mM

100ul

25uM

1. Condition the medium without B27 in glial cultures (80% confluent) for 24 hrs.
2. Add B27, filter, store at 4 degrees C till needed.

CMF Buffer

Ingredient

Stock Conc.

Amount

Final Conc.

H2O (Sterile)



45ml



PBS

10x

5ml

1X

Glucose (sterile)

20%

0.25ml

1%

Fungizone

250ug/ml

0.5ml

2.50ug/ml

Gentamicin

10mg/ml

0.25ml

0.05mg/ml

Filter and store at 4 degrees C.

Cysteine Water

Ingredient

Stock Conc.

Amount

Final Conc.

H2O (Sterile)



7.35ml



Cysteine

0.02M(15.7mg/5ml)

0.5ml

1.25mM

CaCl2

0.5M(736mg/10ml)

30ul

1.90mM

Glia Media (10% FBS)

Ingredient

Stock Conc.

Amount

Final Conc.

MEM



45ml



FBS



5ml

10%

Glutamine

20mM

500ul

2mM

Glucose

20%

845ul

0.34%

Gentamicin

10mg/ml

250ul

0.5mg/ml

Insulin

25mg/ml

20ul

0.01mg/ml

Papain Enzyme Solution

Ingredient

Stock Conc.

Amount

Final Conc.

Cysteine Water

1.25mM

2ml

1mM

Papain suspension

1151unit/ml (Sonicate for 15', vortex for 5')

43ul

20unit/ml

Hank's Balanced Salt Solution

5X

0.5ml

1X

Kynurenic Acid

0.5M

2.5ul

0.5mM

HCl

3N

Varies (around 5ul)

varies

The pH should be adjusted with HCl to 7.3. Then filter.
It takes about 30-60 min at room temperature to activate the papain.

Hank's Balanced Salt Solution 5X
(Modified) 50ml

Ingredient

Stock Conc.

Amount

Final Conc.

NaCl

5M

5.8ml

580mM

KCl

1M

1.35ml

27mM

NaHCO3

0.5M

13ml

130mM

NaH2PO4

1M

0.5ml

10mM

MgSO4

0.5M

0.5ml

50mM

Glucose

20%

5.625ml

2.25%

H2O



23.225ml



Filter and store at 4 degrees C.

Kynurenic acid (0.5M)
1mg KA
10.57ul 1N NaOH
Poly-d-Lysine solution (0.1 mg/ml)
5 mg Poly-d-Lysine
50ml ddH2O
store at -20°C in dark.

Stock Solution Preparation

L-Glutamic acid (2.5mM)
20mg L-Glutamic acid
54.4ml ddH2O
DNAse (stock conc. 2.0mg/ml)
10ml 1X PBS
20mg DNAse.

Make 1ml aliquots of the above stock solutions.
Make 1ml aliquots of Glutamine, B27, fungizone, and 200ul aliquots of insulin
Store at -20°C

SIGMA CATALOGUE NUMBERS:
L-Glutamic Acid G1251 MW 147.1
Cysteine C-1276 MW 157.6
Polysine-D-lysine P-7280 MW 30,000-70,000
Insulin I-5500 MW 5733
Kynurenic acid K-3375 MW 189.2
Hank's Balanced salt solution H-8389

GIBCO CATALOGUE NUMBERS:
MEM 11090-081
NeuroBasal Media 21103-049
B27 17504-044
Gentamicin 15710-064 (10mg/ml)
L-Glutamine 25030-081 (200mM)
Fungizone 15290-018 (250ug/ml)

WORTHINGTON BIOCHEMICAL CORP:
PAP Papain suspension LS003126 (52.1mg/ml, 22.1u/mg)
DNAse I LS002006 (3.017u/mg)
Ted Pella, Inc. :
wax sheets #109 (1lb. box)

Coronal Plate 5
 

Coronal Plate 7
 

Coronal Plate 8
 

 

GLIA CELL CULTURE PROTOCOL

 

 

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

 

 

Ingredient

Stock Conc.

 

Amount

 

Final Conc.

 

NaCl

 

5M

 

5.8 ml

 

580mM

 

KCl

 

1M

 

1.35 ml

 

27mM

 

NaHCO3

 

0.5M

 

13 ml

 

130mM

 

NaH2PO4

 

1M

 

0.5 ml

 

10mM

 

MgSO4

 

0.5M

 

0.5 ml

 

50mM

 

Glucose

 

20%

 

5.625 ml

 

2.25%

 

H2O

 



23.225 ml

 



Filter and store at 4°C

6) Cysteine Water

 

 

Ingredient

Stock Conc.

 

Amount

 

Final Conc.

 

Sterile H2O

 



7.35ml

 



Cysteine

 

0.02M(15.7mg/5ml)

 

0.5ml

 

1.25mM

 

CaCl2

 

0.5M(736mg/10ml)

 

30ul

 

1.90mM

 

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)

 

 

  

  

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