Culturing neurons (based on Gavin Rumbaughs protocol modified by Richard Cho and

Jason Shepherd)

Coverslip Preparation

18 mm Coverslips #0 Thickness aka German coverslips (Assitent 97647 Sondheim,
Germany distributed by Carolina Biological AA 63 3013). Try not to use any other
brand, they simply dont work as well.
Poly-L-Lysine (Sigma P-2636)
Nitric Acid (reuse <6 times)
Trizma buffer pH 8.5 0.1 M (Sigma T1194) filter sterilized
MilliQ Water (sterile)
Be sure to use ethanol resistant materials for culturing protocols

Day 1 (>4 days prior to dissections)
*18mm cover slips: 12 well plate (staining)*
*25mm cover slips: 6 well plate (live imaging)*
-use detergent-free materials from the Live Cell Culturing box, do not put trays or
anything on bench, place clean paper towel down first
1. Incubate slides in Nitric acid (O/N)
a) Load cover slips with forceps into porcelain tray (2 slips/slot)
i) 23 covers/tray: 1 cover straight, 1 diagonally
1. *1 rack: 2, 12 well plates*
b) Use clamp (at Chris bench) to place racks carefully into glass tray of N.A. in the
hood (closest to pcs)
i) Close the tray lid, close the hood door; change acid every 2 months

Day 2-4 (>3 days prior to dissections)

1) Rinse slides and trays in bucket with 1L MilliQ water on rotary shaker (or if shaker is
broken, use stir bar in bucket on stir plate)
-use MilliQ water on later rinses
a) 1 rinse, 2 washes x 1 hour, 1 O/N wash; put DI in bucket and rinse the trays for the rinse
step, before first actual wash, be sure to rinse the bucket with water to remove excess
acid
b) 1 rinse, 2 washes x 1 hour
2) Dehydrate slides with 100% EtOH washes. (EtOH from pump in radioactive room)
a) Wash trays containing slips in bucket containing EtOH, enough to cover the trays
b) Take trays out of EtOH and wrap in aluminum foil
c) Bake coverslips in the foil for 2 hours @ 200F, oven by stereotax and microwave, set to
120 min at 180F
*Coverslips can be stored for weeks*

Poly-L-Lysine (Sigma P-2636)
-1 12 well plate uses 6ml solution/plate (0.5ml per well); make 15ml total
Located: Monicas freezer (in tupperwear with crystals)
(1) Carefully put PLL in conical using two spatulas and weigh
(2) Add Tris to volume (ie/ 0.02g PLL + 20mL Tris)
(3) Pass the mixed solution through a filter (0.2uM filter attached to a syringe large
enough to hold all the PLL + Tris) into a new conical; on coating day, pass
through filter under hood to make sure solution is sterile
(4) Separate PLL into aliquots and freeze

3) COATING: Add PLL to slips (Sigma P-2636; 1 mg/ml in Trizma buffer pH 8.5 0.1 M
(Sigma T1194)). Transfer slips from the trays to plates with the cleanest, most exposed side
facing up using heat-sterilized foreceps
*Be sure dishes and PLL/Tris solution are @ same temperature (ie/ both pre-warmed to
37C) to prevent coverslips from floating.
*Want NO BUBBLES under cover slips
*PL: 20x stock in freezer of tissue culture room (left)
*Place slips into dish (clean side up) with sterilized foreceps- using flame
a) Add either 1mL PLL to each slip in 6-well plate (1mL) or
0.5mL PLL to each slip in 12-well plate (0.5 mL)
b) Incubate plates in PLL O/N @ 37C incubator (2 days before dissection)
c) Rinse PLL, wash 2x 10min, wash O/N (1 day before dissection)

\
-----------------------------------------------------------------------------------------------------------
Day 5 (dissection + plating day)
*Put NM5 into 37C water bath

Sometime before dissections:

Make Reagents

B27 supplements (Gibco 17504044)- Freshly added, from Left Culture Room Freezer
Papain (Worthington LS003119, dilute to 20 mg/ml in dissection media for stock. This
will be a slurry. Store in single use aliquots. Freeze thaw no more than twice. Must be
heat activated at 37C for 30 minutes before use. Be sure to mix well before use)- freeze
until dissection day, is similar to trypsin used in mammalian cell culture; 6.5 papain- for
6 rxns (25.3 u/mgP) and want ~20units/rxn
Characterized Horse Serum / Hyclone / SH30074.03- normally in left freezer culture
room from 500ml stock solution
10x HBSS (w/o Ca and Mg; Gibco 14185-052)
Pen/strep (Gibco: 15140122)
Pyruvate Gibco: 11360070
Hepes Gibco: 15630080
1 M Glucose (Filter Sterilized)
Ara C (Sigma C-6645, for 1000X stock make 2.5 mM solution in ddH
2
0, store frozen)
DNase (Sigma DN-25, make 1 % stock solution in dissection media)
70 uM cell strainer
Neurobasal Media (Gibco 21103-049)
Use ONLY Corning brand dishes. Other dishes (e.g. Falcon) result in uneven distribution
of neurons (aka crop circles).

Make Dissection media (DM): 6 rat pup brains (5 plates)
50 ml 10x HBSS (w/o Ca and Mg; Gibco 14185-052): cold room
5 ml pen/strep (Gibco: 15140122): tissue culture room left freezer left door top shelf;
aliquots of 5ml located in culture room fridge
5 ml pyruvate (Gibco: 11360070): cold room
5 ml Hepes (Gibco 15630080) 10 mM Final: cold room
15 ml Glucose 30 mM Final (1M stock): cold room
420 ml Mill-Q water

Make Neuronal Media 5 (NM5): 6 rat pup brains (5 plates)
*make NM5 + B27 (keep in 37C bath) before doing dissection*

230 ml Neurobasal: cold room, green storage container on floor
12.5 ml HS: tissue culture room left freezer left side
2.5 ml pen/strep
5 ml Glutamax 1: tissue culture room, glass door cabinets @ RT
On of day use add 5mL 2% B27 supplement
Method:
(1) Set up filter (do not attach to aspirator hose)
(2) Pour a little media into filter first
(3) Add HS, B27, pen/strep, Glutamax
(4) Add media to bring up to total volume of 250mL
(5) Attach hose to white adaptor to filter, turn on vacuum

Rinse coverslips
1) Rinse off PLL from coverslips in wells
*Wash with 1mL diH2O*
a) 1 rinse, 2x10min washes + 30 min wash + 2x 10 min washes
*Use new HEAT STERILIZED tips for each aspiration, tip plates when aspirate,
carefully add water into a clean conical and then into wells by tipping water slowly out of
conical (half full well), mark glove for each wash *
2) Pipette ~0.5mL NM5 into each well and store plates in 37C incubator.

Dissection

3) Put 2, 10cm plates on ice for use as dissection dishes (add ice cold DM)
4) Put 1, 15mL conical vial on ice for hippocampi (add 2mL ice cold DM)
5) Sterilize tools in 70 % EtOH (soak in EtOH then let air dry)
6) Pre-warm papain and DNAse to 37C (water bath)- from Left Fridge in culture room
7) Extract the hippocampi
a) Prepare dissection area
(1) Set out diaper, tools, dishes to collect pups in
b) Euthanize mother
(1) Place rat into plastic box
(2) Close lid
(3) Turn on CO2 for >2minutes
(4) Dislocate spine to make sure rat is dead
c) Remove pups from animal.
*It is important to do this quickly after the mother rat has been killed*
(1) Spray down fur with EtOH
(2) Cut up belly (pinch with blunt forceps and cut through fur and skin)
(3) Pull out pups and cut out of mother rat (huge pearl necklace)
(4) Place pups in dish then dish under hood
(5) EtOH clean all tools used
d) Remove hippocampi and place in 15 ml conical.
*Note, blood is toxic to the brain so remove brain to fresh dish immediately*
(1) Separate sacks from each other and from umbilical cord
(2) On a tissue in a dish: Cut pups out of sacks
(3) With forceps hold down the pups body and use other forceps to cut through
membrane on head then through the skull to expose the brain (peel everything
back), open the forceps onto either side of the brain, pinch them a little
underneath the brain and pull the forceps up to pick up the brain and place into
separate dish with DM in it.
e) Remove hemispheres
Cut 1: between the cerebrum and the cerebellum
Cut 2: down the middle of the brain through the corpus callosum
Cut 3: remove the basal brain (pinch in with one set of forceps and use the 2
nd

pair to slice it off by swiping the 2
nd
pair down the 1st pair quickly)
f) Place medial side down
g) Remove olfactory bulb
Cut 4: Hold the cerebrum with 1
st
pair and olfactory bulb with the 2
nd
. Pull the
two apart
h) Remove meninges by sliding tweezers in hole left by olfactory bulb
i) Peel hippocampicampus away from cortex (looks like a banana)
Cut 5: Cut hippocampi out of cerebrum and use forceps to pull it out
j) Put hippocampicampus into the conical on ice.

Plating

8) Tranfer hippocampi
a) Pipet hippocampi plus 2mL DM to a fresh conical
9) Digest
a) Add 20 ul papain (mix before use to mix precipitate in) + 5 l DNase (Final [.01 %]) to 2
ml DM/hippocampi
b) Cap, invert and incubate in water bath (37C)
c) Mix every 5 min for 20 min total
10) During digestion wait period: Prepare dissociation pipets by using flame under hood, be
gentle, make each the diameter of the previous tip
-fire polish Pasteur pipette to the size of the original diameter
*You want 1 small, 1 smaller and 1 smallest pipet tip*
a) Twist tips of glass pipets in a flame to shrink the diameter of the pipette tips
11) During digestion wait period: Prepare plates
a) Take out plates from incubator and aspirate off the solution.
b) To each well add ~1mL NM5 to stop papains reaction
c) Store plates in 37C incubator until digest finishes
12) After digest finishes: Dissociate the hippocampi using 3 fire polished Pasteur pipets with
sequentially smaller tip diameters.
*Titrate the hippocampi through a fire-polished pipette starting with the largest pipette*
a) Discard the papain solution the hippocampi are in (with pipette + pipette man)
b) Wash hippocampi 2 x 5mL with NM5
**To stop the digest reaction: over saturating protein with serum present in the
NM5-NM5 has/is media)**
c) Add 2mL NM5 to hippocampi (should have 2mL total now)
d) GENTLY triturate 5-6xs solution, shooting tissue against wall of tube.
**Prevent bubble formation. Neurons trapped in bubbles will die**
e) Repeat d with smaller and smaller diameter glass pipette tips
13-17 only for CORI TCAL NEURONS
13) Dilute cell mixture with 10mL NM5 before straining
14) Run suspended cells through a 70 uM cell strainer.
a) Run cells through strainer
b) Add 5mL NM5 to sides of conical to capture remaining hippocampi cells and run this
through strainer too.
15) Spin down cells @ 1,400RPM (says 1.4) for 4.5min
16) Take off and discard NM5 from cells (do not disturb pellet of cells)
17) Re-suspend cells in 5mL NM5
18) Count cells with hemocytometer.
*Only count phase-bright cells. Dont forget to factor in dilution for final cell concentration.
Should get around 1.0 x 10
6
neurons per rat pup (half that for mice).*
i-v only for CORI TCAL NEURONS; for HI PPOCAMPAL, just add 10ul straight into
hemacytometer
i) Cut the tip of a yellow pipet tip
ii) Pipet 80uL NM5 and 20 uL cell suspension into a test tube (1:5 dilution)
iii) Pipet 10uL cells into hemocytometer (< end)
iv) Count cells under a microscope in the 4x4 box (all 16 squares worth of cells).
v) Calculate number of total cells: (# cells counted in 4x4 box of hemocytometer) x
(dilution factor 5) x (total volume of suspended cells in conical)
19) Load cells onto coverslips
1.0 x 10
6
/well for 6 well dish (1 ml).
0.5 x 10
6
/dish for coverslips (3 ml).
Want 3.5 x 10^4 cells/cm2 (for staining)
Want 7.5 x 10^4 cells/cm2 (for live imaging)
12 well dishes = 4cm2/well
6 well dishes = 10cm2/well
Calculate how much sup to add per well
20) Add sup to each well.
a) Label plates (hippocampus, # cells counted in hemocytometer, G for glass coverslips in
each well, date, initials,
b) Add _______miroL to each well
c) Shake stacked plates to evenly distribute cells.
d) Place plates in 37C incubator
e) Shake plates again when in incubator
21) After 2 hours, look at cells and determine if they have attached.
22) Once cells are attached, change media to fresh NM5.
*1mL for 12 well dish
SKIP 23-25 for HIPPOCAMPAL NEURONS
23) Check cells daily: Add AraC when glia are about 30% confluent (~ 3-4 days). DO NOT add
AraC directly to media. Remove of media and mix in 2X AraC before replacing.
24) On DIV4 replace media (remove 1.5 ml, add 2 ml) with Glia Conditioned NM1 ( Add B27
right before feeding glia conditioned NM1 to Neurons).
25) Cultures are then fed with media changes every 3
rd
day (remove 1.5 ml and add 2 ml) to
protect against media evaporation and metabolic byproduct accumulation.

For DIV 4 and older, neurons should be fed with NM1 that has been Glial conditioned
overnight. See below for preparation of Glia
------------------------------------------------------------------------------------------------------------

Neuronal Media 1 (NM1)
240 ml Neurobasal
2.5 ml HS
2.5 ml pen/strep
2.5 ml Glutamax 1
On day of use add 2% B27 supplement
Preparation of Glia for Neuron Feeding

Reagents

Hanks buffered saline solution (HBSS) / Invitrogen / 14170-112
P/S/G Invitrogen / 10378-016
2.5% Trypsin / Invitrogen / 15090-046
Mem / Mediatech / 15-010-CV
Horse Serum / Hyclone / SH30074.03
DNase / Sigma / DN-25
40 um Filter / Falcon / 352340
1X Trypsin (check
Collagen: http://www.inamedbiomaterials.com: PureCol 2586-C01-0704
Large 75 flasks

Reagents and Media Preparation

1. Dnase:
100 mg Dnase
10 ml HBSS
Filter Sterilization
Store at -20
o
C in 1ml and 0.5 ml Aliquots

2. 10% Glia Media
500 ml MEM
55 ml Horse Serum
5 ml Pen/Strep/Glutamine

3. Collagen Coating
Dilute Collagen with water 1:2 (e.g. 1 ml of Col and 2 ml of H
2
O)

Coating Collagen Plates

Preparation
** Only use Corning Brand 60 mm dishes. DO NOT USE ANY OTHER BRAND.
1. Collect 6 X medium flasks
2. Prepare Collagen Mixture (1 part collagen: 2 parts H
2
O). 10 ml total is enough.

Plating
1. Flash coat the flasks and plates by adding collagen mixture and swirling to completely
coat plastic.
2. Remove collagen and reuse on next flask or plate until all flasks/plates have been flash
coated.
3. Remove flash caps and plate tops and allow to dry O/N in hood.
4. The next morning elevate plates and flasks and treat with UV for 20-30 minutes.
5. Replace caps and tops and wrap in aluminum foil to protect from light.
6. Treated flasks and plates can be stored at 4
o
for 2-4 weeks.


A. Glia Cell Culture

1. Postnatal P1-3 day rats
2. Soak dissection instruments in 70% ethanol for 30 min before dissection.
3. Warm 10% Glia Medium to 37
o
C in water bath.
4. Warm up DNase (1 ml and 0.5 ml tube) and Trypsin (2X 1ml tube) at RT.

B. Dissection (Keep all dissected tissue on ice)

1. Remove postnatal head into 60 mm Petri Dish with 2 ml HBSS
2. Remove brain stem, cerebellum, and diencephalons.
3. Peel off meningis and put cortex into a 60 mm dish with 4 ml HBSS on ice.
4. 1-2 pups should be enough.
5. After dissection, chop cortex into small pieces.
ALL dissection should be done under a dissection uscope.
Dissection should be performed as quickly as possible.
Keep all dissected tissue on ice as much as possible.

C. Digestion:

1. Transfer the chopped tissue in 4 ml HBSS into 15 ml conical Tube.
2. Add another 4 ml of HBSS into the above tube. The total volume should be 8 ml.
3. Add 1 ml of 2.5 % Trypsin and 1 ml Dnase into the tube, the total volume should be 10
ml, then mix.
4. Incubate at 37
o
C water bath for 15 min, mix every 5 min.
5. Transfer supernatant into a 50 ml conical tube (about 8 ml).
6. Add equal volume of 10% Glia Medium to block digestion
7. Add 5 ml HBSS, 0.5ml DNase, and 1 ml 2.5 % trypsin into 15 ml conical tube containing
remaining digested tissue.
8. Incubate at 37
o
C water bath for 15 min, mix every 5 min.
9. Transfer the supernatant into the 50 ml conical tube (Step 5).
10. Add equal volume 10% Glia Medium into the 50 ml conical tube to block digestion.
11. Filter the cell suspension with a 70 um filter into another 50 ml conical tube.
12. Centrifuge at RT for 5 min, set speed at 3.


Plating Cells:
Day 1:
.
1. Discard supernatant carefully.
2. Resuspend cell pellet in 4 ml 10% Glia Medium, and mix well.
3. Count cells and calculate density. Use Trypan Blue staining to count live cells. 4 parts
stain: 1 part cells. Count only clear cells. Optional
4. Plate 2x10
6
cells per collagen treated 75cm
3
flask (PLATE 2X FLASKS PER
DISSECTION) (Vol = ~15 ml).

Day 2-4:

1. Before feeding, knock the flask firmly (almost vigorously) with the palm of your hand 3-
4 times. This will knock off unwanted cells (uglia, neurons, etc).
2. Feed Cells daily with 10% Glia Medium.

Day 7 (or when confluent):

1. Remove media from flask and wash with PBS.
2. Add 2.5 ml of TE.
3. Resuspend in 10% Glia Midium and spin down 5 minutes at setting 2.
4. Remove media. Be careful, pellet will be small.
5. Resuspend in 10 ml of 10% Glia Medium
6. Split into however many flasks are needed. Repeat knocking and feeding until confluent.

Day 14-18:
1. Cells are ready to use. Optional: add AraC as described in neuron section to inhibit
growth. However, cells typically stop dividing due to contact inhibition.
2. Glia are good for 5-6 weeks with regular conditioning of neurons. It takes ~ 2 weeks
after dissection for glia to be ready for conditioning so plan accordingly.

High-Density cortical Neuron transfection protocol

For 1 well/12 well plate; however, this protocol scales linearly with the number of transfected
coverslips. Just multiply each reagent by the number of coverslips.

1. DMEM (w/o glutamine but with GlutaMax) should be freshly buffered with HEPES
before each transfection (h-DMEM).
a. Take small amount of DMEM, warm to 37C, then add 10 mM HEPES (100x)
2. Add 50 ul h-DMEM (0.5-3ug) to a ufuge tube, then add 3 ul of shaken Lipo2k (Tube A).
a. You must allow diluted lipo2K to sit for at least 2 min, but no longer than 10 min.
3. Take h-DMEM and dilute DNA (0.5-3 ug) to 50 ul (in a separate tube; Tube B)
1.5 ug DNA/3 ul of Lipo2k
4. Combine contents of A and B. Pipette up and down vigorously (or vortex) for 10
seconds followed by a quick-spin (Tube AB).
5. Incubate Tube AB at RT (with cap closed) for 15 min.
a. During this incubation period, retrieve one 12-well plate and neurons to be
transfected.
b. 5 minutes before the incubation time has expired, place 700 ul of NIM (+B27)
into the 12-well plate
c. Remove a coverslip from the dish and place into a well containing NIM
d. Place plate into incubator without CO2
6. Once the 15-minute incubation period has expired, add contents of Tube AB (100 ul) to
well containing NIM and the coverslip in a drop wise manner
a. Gently shake the plate to disperse the DNA precipitates.
7. Incubate for 1 hr at 37C without CO2
a. NOTE: transfection can proceed up to 3 hrs, but I recommend increasing DNA
concentration before lengthening the incubation time.
8. After the incubation time has expired, simply switch the coverslip from NIM to a well
containing previous conditioned growth media.
9. One can analyze transfection efficiency 12-72 hours later. However, longer post-
transfection periods result in significantly increased toxicity to transfected neurons.


















Neuronal Incubation Media (NIM)

82 ml ddH
2
O
10 ml 10x MEM (Invitrogen 11430-030)
2 ml Glutamax (Invitrogen)
1.5 ml 1M HEPES (Invitrogen 15630-080)
1 ml 100 mM Sodium Pyruvate (11360-070)
3.3 ml 1M Glucose
Ph 7.4 filter

1x 2x 3x 4x 5x
H2O 82 164 246 328 410
10X MEM 10 20 30 40 50
Glutamax 2 4 6 8 10
Hepes 1.5 3 4.5 6 7.5
Sodium
Pyruvate 1 2 3 4 5
3.3 Glucose 3.3 6.6 9.9 13.2 16.5

Troubleshooting Neurons

Culturing neurons can be technically challenging. Failure to properly execute each step outlined
above can have disastrous effects on the health and utility of your neurons. Therefore, at least
initially, it is important to take great care in following the protocol.

If you do run into difficulties, identifying your problem will be challenging. If neurons suddenly
stop working, often times, its more cost effective and efficient to replace all reagents and start
with fresh reagents (e.g. B27 and serum).

Below are some common pitfalls and possible solutions. Be careful, sometimes the most
obvious things are over looked: CO2 calibration, water in the incubator, correct temperature,
incomplete washing after substrate (excess PLL is toxic to neurons), etc

Cells not sticking to plate - Possibly substrate problem. Order new substrate and
make sure to properly store PLL (desiccate and bring to
room temp before opening).
- Instead of 1 2 hours after dissection, you can replace
NM5 after O/N incubation
Neurons die in <1 weeks - Make sure you are not over-plating your neurons. Try
serial dilutions
- May have a substrate problem. Is there clumping of
cell bodies and fasciculation of processes? These are
signs of substrate problem. See above for substrate
instructions.
- Make sure glia are growing to around 30-40%.
Sometimes, AraC is added too early.
- Make sure glia plates are pure. That is, it should be free
of uglia. These cells can secrete factors that can
adversely affect the health of your neurons. Be sure to
practice vigorous knocking of glia flasks during glia
preparation.
Clumping of cell bodies and
fasciculation of processes
- New substrate.
- Make sure to incubate in NM5 O/N after washing
substrate.
Cells die after transfection - Use less lipo2k or DNA
- Decrease expression time
- Make sure you dont manipulate the cells on the days of
feeding. After feeding, the cells can be especially
fragile. Wait at least O/N.