Neuronal Culture Protocol (Originally from the Worley’s lab)
Coverslip Preparation
14-15-18 mm Coverslips #0 Thickness (Carolina Biological AA 63 3013)
Poly-L-Lysine (Sigma P-2636)
Nitric Acid
Borate Buffer (100 mM Na2B4O7*10H2O: 19.1 g in 500 ml H20, pH 8.5 filter sterilized)
MilliQ Water
Day 1
1.
Day 2
1.
Day 4
1.
2.
Incubate coverslips in MilliQ water overnight
Incubate in Nitric acid for two days
Rinse with MilliQ (2L per rack) water 4x 1-2hr on rotary shaker
Dehydrate with 2X 100% EtOH washes. Coverslips can be stored in EtOH for
weeks.
3. Add PLL (Sigma P-2636; 0.02 mg/ml in borate buffer 0.1 M; pH 8.5; filter
sterilize) to either 60 mm dishes (3 ml) or to 12 sterilized coverslips in 12-well
plate (1 ml). Be sure dishes and solutions are pre-warmed to 37C to prevent
coverslips from floating.
4. Flame coverslips and place in 6 well dishes (where neurons grow at best) &
Incubate O/N 37C.
Day 5 Before plating neurons
1. Rinse PLL with 3 water rinses.
2. Add NM5 media and then add dissociated neurons.
3.
Neuronal Dissection and Plating
Reagents
1.
2.
3.
4.
5.
6.
7.
8.
9.
B27 supplements (Gibco 17504044) or Neuromix (F01-002, PAA).
Papain (Worthington LS003119) or Accutase (L11-007, PAA)
FBS
10x HBSS ( can make it urself with MilliQ water)
Pen/strep
Pyruvate
Hepes
1 M Glucose (Filter Sterilized)
Ara C (Sigma C-6645, for 1000X stock make 2.5 mM solution in ddH20, store
frozen)
10. Neurobasal Media or Neuronal Base Medium P (U15-059, PAA)
11. DNase (Sigma DN-25, make 1 % stock solution in dissection media)
12. 40 uM cell strainer
Dissection media (DM)
50 ml 10x HBSS
5 ml pen/strep 5 ml pyruvate
5 ml Hepes 10 mM Final
15 ml Glucose 30 mM Final (1M stock)
420 ml Mill-Q
Neuronal Media 5 (NM5)
230 ml Neurobasal or Neuronal Base Medium P (U15-059, PAA)
12.5 ml FBS
2.5 ml pen/strep
5 ml Glutamax 1
On of day use add
2% B27 supplement
Dissection
1.
2.
3.
4.
Prepare ice cold 10 cm plates with DM for use as dissection dishes
Prepare ice cold 15 ml conical with 2 mls DM
Sterilize tools in 70 % EtOH
The hippocampus can be obtained form E16 embryo or P0 pups, depending on
the experiment, E16 culture give an almost pure neuronal culture whereas P0
neuronal cultures give a mixture of glia and neurons…but in both case the cells if
gently cared for look GOOD!
5. Procedure:
a. Remove hippocampi and place in 15 ml conical Note, blood is toxic to the
brain so remove brain to fresh dish immediately after removal from skull
b. Remove hemispheres
c. Place medial side down
d. Remove olfactory bulb
e. Remove meninges by sliding tweezers in hole left by olfactory bulb
f. Peel hippocampus away from cortex
6. Add 50 ul of papain or 1;1 Accutase and 20 µl of DNase (Final concentration of
.01 %) to 2 ml of dissection media. Incubate in water bath (37C) with gentle
perturbation every 5 minutes for 30 min.
7. Warm 50 mls of NM5 to 37C and thaw B27 or Neuromix.
8. Prepare dissociation pipets. Use 3 fire polished pasteur pipets with sequentially
smaller tip diameters.
9. Aspirate the solution and add 2 ml of pre-warmed NM5 with freshly added B27.
10. Dissociate the tissue by gently triturating the hippocampi through a fire-polished
Pasteur pipette. Starting with the largest pipet:
a. GENTLY triturate twice, shooting tissue against wall of tube. This will
prevent bubble formation. Neurons trapped in bubbles will die.
b. Let tissue settle until large chucks are on bottom (~1 minute)
c. Remove supernatant to fresh tube
d. Gently add 2 mls NM5 and go back to step (a) with smaller pipet
11. Dilute cell mixture to 10 ml with plating media and then run through a 40 uM cell
strainer.
12. Spin cells down at 3000 G (2000 rpm) for five minutes.
13. Re-suspend cells in NM5 + B27. Count with hemocytometer. Only count phasebright cells. Should get around 1.0 x 106 neurons per rat pup (half that for mice).
For a 6 cm dish, add 150-500K cells in 3 mls.
14. After 1 hour, look at cells and determine if they have attached. .
15. Check cells daily. Add AraC when glia are about 30% confluent (3rd-4th day).
Refresh with ½ of the media containing 2X AraC.
16. Cultures are then fed with ½ media changes every 3rd day (remove 1.5 ml and
add 2 ml) to protect against media evaporation and metabolic byproduct
accumulation. The media should be conditioned on glia o/n to provide
growth factors essential for the neurons to survive.