Preparation of Rat Dorsal Root Ganglion Neurons
Materials
• one timed pregnant (E16) Sprague-Dawley rat (total ~10 embryos) or E14.5 mouse
pregnant
• Leibovitz’s L-15 medium (L-15, Gibco 11415-064)
• Dulbecco’s phosphate-buffered saline (D-PBS, Gibco 14190-144)
• sterile distilled water (dH2O, Gibco 15230-162)
• 0.25% trypsin solution (Gibco 25200-056)
• fetal bovine serum (FBS, Gibco 16000-044)
• Hanks’ balanced saline solution (HBSS, Gibco 14170-112)
• blunt forceps and rat-tooth “pick-up” forceps
• 2 small- and 1 medium-size dissecting scissors
• micro-dissecting scissors (Biomedical Research Instruments 11-1350)
• #5 dissecting forceps (Roboz RS-5045)
• 4-well tissue culture dishes (Nunc 176740)
• 12-mm German glass coverslips (Carolina Biological Supply KS-63-3029): place in
large glass Petri dish and dry-autoclave to sterilize
• 245 x 245 mm square tissue culture trays (Corning 431272): clean by wiping down
with 70% ethanol and exposing to UV light 30 minutes prior to use
• 2x2 gauze pads
• collagen (Cultrex Trevigen 3440-100-01)
• concentrated ammonium hydroxide solution (NH4OH)
or
• poly-L-lysine (PLL, Sigma P-1274)
• laminin (1.25 mg/mL, Collaborative Biomedical Technologies 40239)
or
• Matrigel, growth factor reduced (BD Biosciences/Clontech 356230)
Preparation (Day 0)
1. Using sterile technique, place autoclaved coverslips in 4-well dishes (15-20 dishes per
dissection).
2. If coating coverslips with Matrigel, thaw solution overnight at 4°C.
Preparation (Day 1)
1. Prior to dissection, soak all instruments in 70% ethanol.
2. Coat coverslips (approx. 60-80 coverslips/15-20 4-well dishes per dissection) with
collagen, poly-L-lysine/laminin, or Matrigel as follows:
Collagen
1. Dilute collagen stock solution 1:10 or 1:5 (if the collagen has been kept at 4C for more
than 2 months) with (glacial acetic acid diluted 1:1000 in sterile dH2O) and mix thoroughly
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by pipeting up/down prior to use (final conc. 50 µg/mL). Make 150ul solution per coverslip
(e.g., 0.5 mL collagen stock + 4.5 mL dH2O for 20 dishes/80 coverslips).
2. With sterile transfer pipet, drip collagen solution onto center of coverslip until covered,
taking care not to apply collagen to plastic dish.
3. After all coverslips are coated, aspirate collagen back into pipet to leave behind a thin
film of collagen.
4. Once all coverslips are coated, place one 4-well dish containing coated coverslips in each
corner of 245 x 245 mm culture trays (you can do that at the very beginning) and lay three
2x2 gauze pads in center of each tray.
5. Wet gauze pads with 1 mL NH4OH and cover trays for 15 min.
6. Remove gauze pads, tilt dish lids with open side facing the front of the hood, and allow to
dry until ready to use.
Poly-L-lysine/laminin
1. Thaw laminin on ice one hour prior to use (store 1.0-mL aliquots at –80°C for up to 3
months after initial thawing).
2. Dissolve 0.25 g PLL in 50 mL sterile dH2O, filter thru 0.22 µm filter, store 1.0-mL
aliquots at –20°C.
3. Dilute PLL 1:10 with D-PBS (e.g., 1 mL PLL + 9 mL dH2O) and add 100 µL to each
coverslip.
4. Let sit at room temperature for 30 min.
5. Wash coverslips 2 x 100 µL sterile dH2O.
6. Dilute 80 µL laminin in 10 mL sterile dH2O and add 100 µL to each coverslip.
7. Let sit at room temperature for 1 hr.
8. Wash coverslips 2 x 100 µL sterile dH2O, then once with medium prior to use.
Matrigel (for use with rat and mouse cultures)
1. Dilute thawed Matrigel (overnight at 4°C) 1:10 with ice-cold DMEM and keep solution
on ice thereafter. Make 1 mL solution per 4 dishes/16 coverslips.
2. Add 150 µL diluted Matrigel each to 4 coverslips and then quickly remove solution to
leave a thin layer of matrix behind.
3. Repeat for each coverslip (re-use Matrigel from previous coverslips), then place one 4well dish in each corner of a 245 x 245 mm culture tray.
Dissection and Isolation of DRGs (Day 1)
1. Sacrifice mother rat by CO2 asphyxiation or dry ice asphyxiation.
2. Place rat in supine position on blue diaper and thoroughly spray abdomen with 70%
ethanol.
3. Using “pick-up” forceps, grasp lower abdominal skin at midline and lift
4. Cut skin with medium-size scissors in an “I” pattern to expose abdominal muscles
5. Lift up muscles and abdominal wall with pick-up forceps and make transverse incision
with small dissecting scissors taking care not to puncture/cut viscera
6. With blunt forceps, grasp uterus laden with embryos near cervical insertion and gently lift
from peritoneal cavity.
7. Cut away connective tissue and suspensory ligaments and place entire uterus in sterile 10cm tissue culture dish. MOVE NOW IN THE DISSECTION HOOD
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8. Remove embryos from uterus by cutting through tissue surrounding amniotic fluid and
gently squeezing embryo through incision.
9. Place all embryos into another 10-cm dish containing L-15, swirl dish to “clean”
embryos, and divide embryos into 2 10-cm dishes with fresh L-15. Transfer individual
embryos to a 6-cm dish with a thin layer of L-15 to isolate DRGs.
10. Lay embryo on its side and, under a dissecting microscope, cut off the head at the
cervical flexure and the tail just caudal to the hind limbs using micro-dissecting scissors.
11. Remove the ventral (belly) portion of the embryo to isolate the dorsal (back) structures
containing the spinal cord.
12. Position tissue dorsal side down and carefully remove any remaining viscera from the
posterior wall.
13. Place one blade of micro-dissecting scissors between vertebral column and spinal canal
at rostral end and very carefully cut through vertebral column proceeding caudally, then
tease apart the right and left halves of vertebral column to expose spinal cord and DRGs.
14. Gently attempt to lift spinal cord from dorsal structures by grasping cord at rostral end
while carefully “cutting” behind and around DRGs to sever adherent tissues.
15. Once entire spinal cord and attached DRGs can be peeled off underlying tissues, transfer
to clean 6-cm dish containing a thin layer of L-15.
16. After isolating all spinal cords, pluck off DRGs using dissecting forceps and transfer to
another 6-cm dish with L-15. If nerve roots are present, they should be snipped away before
removing DRGs from cords.
Generation of DRG Explant Cultures (Day 1)
1. Add 200 µL C-media per coverslips and swirl the dish to cover the entire surface
2. Using a #5 dissecting forcep, deposit 1 DRG onto the center of each coated coverslip.
3. Grow cells in 37°C incubator with 5% CO2.
(Days 2, 3, and 4)
5. Change supplemented Neurobasal every other day.
(Days 5-25)
6. Change medium to C-medium containing 50 µg/mL vitamin C (ascorbic acid) to promote
myelination by endogenous Schwann cells.
7. Change medium every 2 days until adequate myelination is observed (within 3 weeks).
Preparation of Purified DRG Neuronal Cultures (Day 1)
1. Swirl dish containing DRGs to force them to the center and carefully remove L-15
medium from the periphery of the dish using a sterile transfer pipet.
2. Add 2 mL 0.25% trypsin solution and incubate 45 min at 37°C.
3. Add 1 mL 10% FBS/L-15 and 1 mL FBS to dish and transfer entire 4 mL to sterile 15-mL
centrifuge tube. Wash dish with another 2 mL 10% FBS/L-15 and add to tube.
4. Centrifuge at room temperature 10 min at 700 rpm.
5. Pour off supernatant and re-suspend cell pellet by gentle trituration in 200 µL Cmedium/1.5 DRG (or 200 µL supplemented Neurobasal medium/DRG, see below) using a
10-mL pipet.
6. With a 2-mL pipet, transfer 200 µL cell suspension to the center of each coated coverslip.
7. Incubate cells at 37°C with 5% CO2 overnight.
(Days 2-18)
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8. Cultures should be cycled for 3 weeks between C-medium (or supplemented Neurobasal
medium) and CF-medium (or NBF) (200 µL/coverslip) to kill dividing cells such as
fibroblasts. If cells were isolated on a Tuesday (Day 1), the regimen would be:
Monday
Wednesday
Friday
Week 1
CF/NBF
C/NB
Week 2
CF/NBF
C/NB
CF/NBF
Week 3
C/NB
C/NB
C/NB
*results in faster “clean-up” of dividing cells but reduced myelination later)
Co-Culturing of Purified DRG Neurons and Schwann Cells (Day 19)
1. Wash Schwann cell cultures growing on 10-cm dishes twice with 37°C HBSS.
2. Add 5 mL 0.25% trypsin solution and incubate at 37°C for 2-3 min.
3. Add 1 mL D-medium and agitate plate and/or gently pipet up and down with 5-mL pipet
to loosen cells.
4. Transfer cell suspension to 15-mL centrifuge tube and centrifuge at room temperature 5
min at 1000 rpm.
5. Pour off supernatant and re-suspend cell pellet by gentle trituration in 1 mL C-medium
using a P1000 pipetman.
6. Count cells and dilute with C-medium to final conc 2 x 105 cells /400 µL.
7. Remove DRG medium and add 400 µL cell suspension/well (i.e., 2 x 105 cells/coverslip).
(Day 20)
8. From this day on, we live them in C media.
(Days 23-37)
9. Change to C-medium containing 50 µg/mL ascorbic acid (vitamin C) and continue to
change medium every 2 days for myelination to proceed over the next 1-2 weeks.
Media Formulations
Stock reagents
• ascorbic acid (Sigma A0278) final 50ug/ml
• B27 supplement (50x, Gibco 17504-044)
• Dulbecco’s modified Eagle medium (DMEM, Gibco 11995-065)
• F-12 nutrient mixture (F-12, Gibco 11765-054)
• fetal bovine serum (FBS, Gibco 16000-044)
• FUDR: 12.3 mg fluorodeoxyuridine (FdU, Sigma F-0503) + 12.2 mg uridine (Sigma U3003)/5 mL MEM, sterile filter, dilute 1 mL + 9 mL MEM before use
• D-glucose (Sigma G-5146): 4 g/20 mL MEM, sterile filter
• L-glutamine (200 mM, Gibco 25030-081)
• insulin (Sigma I-6634): dissolve 10 mg in 100 µL 1.0 N HCl, dilute in 20 mL sterile
water, vortex, filter thru 0.22 µm filter, store at 4°C for 4-6 wks
• minimum essential medium (MEM, Gibco 11090-081)
• Neurobasal medium (NB, Gibco 21103-049)
• 2.5S nerve growth factor (NGF, Harlan 005017): 1 mg/2 mL sterile dH2O, store 50-µL
aliquots at –20°C
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• progesterone (Sigma P8783)
• putrescine (Sigma P7505)
• sodium selenite (Sigma S5261)
• transferrin, rat (11 mg/mL, Jackson ImmunoResearch 012-000-050)
C-medium
MEM
D-glucose
FBS
L-glutamine
NGF
870 mL
20 mL (final 4 g/L)
100 mL (final 10%)
10 mL (final 2 mM)
100 µL (final 50 ng/mL)
CF-medium
C-medium containing 1% (v/v) FUDR (final 10 µM FdU and 10 µM uridine)
D-medium
DMEM
FBS
L-glutamine
445 mL
50 mL (final 10%)
5 mL (final 2 mM)
N2 medium (a.k.a. defined medium)
DMEM
F-12
insulin
transferrin
1000x N2 stock
NGF
200 mL
200 mL
2 mL (final 5 µg/mL)
364 µL (final 10 µg/mL)
400 µL
41 µL
1000x N2 stock:
progesterone
putrescine
sodium selenite
final (1x) conc.
6.3 ng/mL (20 nM)
1.6 µg/mL (100 µM)
5.2 ng/mL (30 nM)
1000x stock (50 mL)
500 µL stock (6.29 mg/10 mL ethanol)*
80 mg
2.6 mL stock (1 mg/100 µL 0.1 N
NaOH and 10 mL DMEM)*
Dissolve all of the above in 50 mL DMEM, filter thru 0.22 µm filter, store 500-µL
aliquots at –20°C. *Make fresh each time.
Supplemented NB
NB
475 mL
5
B27 supplement
D-glucose
L-glutamine
NGF
10 mL
10 mL (final 4 g/L)
5 mL (final 2 mM)
50 µL (final 50 ng/mL)
NBF
NB-medium containing 1% (v/v) FUDR (final 10 µM FdU and 10 µM uridine)
We have recently observed that cycling the cultures with NB and NBF media can easily
purify the neurons, whereas for myelination C-medium supplemented with ascorbic acid
still works better.
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