PROTOCOL

Production of dissociated sensory neuron cultures and

considerations for their use in studying neuronal
function and plasticity
Sacha A Malin, Brian M Davis & Derek C Molliver

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, 3350 Terrace Street, Pittsburgh, Pennsylvania 15261, USA.
Correspondence should be addressed to S.A.M. (sam64@pitt.edu).
Published online 15 February 2007; doi:10.1038/nprot.2006.461

Dissociated primary sensory neurons are commonly used to study growth factordependent cell survival, axon outgrowth,
differentiation and basic mechanisms of sensory physiology and pain. Spinal or trigeminal sensory neurons can be collected from
embryos, neonates or adults, treated with enzymes that degrade the extracellular matrix, triturated and grown in defined media with
or without growth factors and additional animal sera. Production of cultures can take as little as 2.5 h. Cells can be used almost
immediately or maintained for as long as 1 month. Ease of production and the ability to control growth conditions make sensory
neuron culture a powerful model system for studying basic neurobiology of central and peripheral nervous systems.

INTRODUCTION
Dissociated rodent sensory neurons are among the most widely specific signaling pathways24,25. Adult and neonatal sensory cultures have been routinely used for physiological studies exploring
used neuronal cell types for primary culture. Sensory neurons are
located in spinal ganglia (also known as dorsal root ganglia, or molecular mechanisms for the detection of physical, chemical and
DRGs) or in cranial sensory ganglia associated with cranial nerves thermal stimuli. In addition, isolated afferents have been used to
study processes important for both central and peripheral nervous
V, VII, VIII, IX and X. The great majority of these neurons are
derived from the neural crest, although some come from placodal system function, such as regeneration26,27 and the role of axonal
tissue (e.g., the nodose ganglion found on cranial nerve X)13. translation of proteins in modulating synaptic function2830.
Sensory neurons can be obtained from DRGs, located in the
intervertebral foramen along the spinal column (see Fig. 1), and Strengths and weaknesses of the model system
from the trigeminal ganglia (TGs) associated with cranial nerve V, A central strength of neuronal culture is tight control of the
neuronal environment, including factors that regulate neuronal
located on the floor of the cranium (see Fig. 2). Proprioceptive
neurons innervating the muscles of mastication are located in the mesencephalic
a
b
c
trigeminal nucleus and can also be cultured4. Both TGs and DRGs can be
L4
T13
removed relatively easily from mouse or
rat embryos shortly after sensory neurons
are generated (approximately on embryonic
day (E) 1013 in mouse5,6 and E914 in
rat79), as well as from neonatal or adult
d
e
f
*
animals. Embryonic and neonatal cells
require trophic factors (especially nerve
T10 T11 T12
growth factor, or NGF) in addition to
T13
L3
L4
L5
*
+
standard culture media to survive; however,
adult cells can be maintained without
trophic factors in defined media with vita- Figure 1 | Dissection of dorsal root ganglia (DRGs) from the mouse and identification of specific ganglia.
In all panels, the head lies to the left. Panels (ac) depict the initial exposure of the spinal cord and the
min supplement (Neurobasal, B27, GLutasegmental distribution of the ganglia within the mouse; panels (df) are magnified views revealing the
MAX, Invitrogen).
location of the ganglia at different segmental levels. Arrows point to ganglia. The first incision is made
Isolated sensory neurons maintain fea- across the spine (a). Next, parallel cuts are made through the vertebrae adjacent to the spinal cord and
tures of in vivo sensory nerve endings10 and overlying muscle and bone are removed (b,c). The last thoracic ganglion (T13) and the ganglion with
have been used in a wide range of develop- the greatest contribution to the sciatic nerve (L4) are identified for orientation with respect to the whole
mental studies, including the investigation mouse (c). Thoracic ganglia are revealed here using pins to pull back the spinal cord (d, not necessary
of programmed cell death1113, growth cone for dissection). The dorsal roots are very short at cervical and upper thoracic levels, and the ganglia lie
closely apposed to the spinal cord. The thoracolumbar transition is easily identified because T13 is just
dynamics1418 and growth factor signaling caudal to the last rib (+ sign) (e). Peripheral nerves are exposed to reveal the ganglia contributing to
pathways1923. Compartmentalized culture the sciatic nerve (f, sciatic nerves labeled with asterisks). Note the large projection from L4, the smaller
systems separating cell bodies and processes projection from L5 and the small branch leaving the L3 nerve to join the sciatic nerve. L4 is generally
have been used to discriminate location- the largest lumbar ganglion.
152 | VOL.2 NO.1 | 2007 | NATURE PROTOCOLS

 2007 Nature Publishing Group http://www.nature.com/natureprotocols

PROTOCOL
function and phenotype in vivo. This precise control of the
extracellular environment is highly advantageous for physiological
and pharmacological studies of the intrinsic electrical properties
of neurons and sensory transduction. Primary sensory neuron
cultures have a number of specific advantages. Sensory ganglion
composition is stereotyped from mouse to mouse; for a particular
ganglion, there is little variation between animals in cell number,
response properties and target innervation. Therefore, the same
population of cells can be readily identified, removed and examined
in parallel experiments from multiple animals. Another advantage
is that each ganglion contains neurons with diverse response
properties, target tissue innervation and growth factor responsiveness3135. For example, some neurons are specialized to detect
noxious stimuli (e.g., acid or tissue-damaging mechanical stress),
whereas others are designed to detect non-noxious stimuli (e.g.,
vibration or limb position). Sensory neurons innervate skin,
muscle, bone and/or viscera, and, importantly, populations of
sensory neurons can be defined and identified by innervation
target. For example, injection of a retrogradely transported dye
into the skin several days before cell isolation allows the identification of cutaneous afferents in culture36. In addition, many neurochemical markers have been identified that distinguish restricted
subsets of sensory neurons37. For example, expression of the
ion channel TRPV1 defines a specific subset of nociceptors, and
the calcium-binding protein parvalbumin is expressed primarily
by proprioceptors38,39.
Isolated sensory neurons are routinely used for sharp electrode
and patch-clamp electrophysiology, as well as calcium imaging.
Sensory neurons can also be grown as cultures with other cell types,
including skin4042 and CNS tissue43,44. Sensory neurons retain the
ability to respond to chemical4547, thermal48,49 and mechanical50
stimuli in culture. As mentioned, neurochemical markers can be
used to infer some aspects of neuronal function, but they do not
unequivocally reveal innervation target (skin, muscle, bone, viscera) or functional modality (e.g., heat, cold, osmolarity or
mechanical responsiveness). Because sensory neurons are specialized to perform specific functions in diverse tissues, interpretation
of physiological and pharmacological data is greatly aided by
knowledge of the innervation target. For example, visceral and
cutaneous sensory neurons have distinct intrinsic electrical properties and diverse evoked responses to ATP and capsaicin5154. These
data suggest that ATP- and capsaicin-sensitive ion channels may

MATERIALS
REAGENTS

. Animals (see REAGENT SETUP)

. 70% ethanol in spray bottle
. Laminin (Sigma, cat. no. L2020)
. Poly-D-lysine (Becton Dickinson, cat. no. 35-4210)
. F12 culture medium (GIBCO, cat. no. 11765)
. FCS (Invitrogen, cat. no. 10082139)
. Papain (Worthington, cat. no. 3126)
. NaHCO3 (Sigma, cat. no. 35761)
. Sterile 0.2 mm syringe filters (Fisher, cat. no. 0975413)
. Collagenase type II (CLS2) (Worthington, cat. no. 4176)
. Dispase type II (MB, cat. no. 165859)
. L-Cys free base (Sigma, cat. no. C7352)
. HBSS without Ca++ /Mg++ (GIBCO, cat. no. 14170)
. NGF (Sigma, cat. no. N6009)
. Penicillin/streptomycin (Invitrogen, cat. no. 15140122)

eye
3
2
4
olf
olf

1
on

tg

eye

Figure 2 | Location of the trigeminal ganglia (tg, double arrow) in the

cranial cavity. on, optic nerves; olf, olfactory bulb. The trigeminal branches
are labeled at the location to be severed for removal of the ganglia:
1, ophthalmic; 2, maxillary; 3, mandibular; 4, fifth cranial nerve.

play a role in visceral sensation distinct from their role in cutaneous

thermal sensation. We and others recommend that studies on
isolated sensory neurons be conducted in retrogradely labeled
(target-identified) afferents whenever possible.
The most significant caveat for primary sensory neuron culture is
that cell isolation requires axotomy of both central and peripheral
processes. This limitation is common to all primary neuronal
culture but is often overlooked in the interpretation of data derived
from dissociated sensory neurons. Thus, sensory neurons in culture
share many of the features of regenerating sensory neurons in vivo.
Isolated sensory neurons robustly express molecules associated
with axonal regeneration, such as GAP43, galanin and plasminogen
activators55. Also, trkA expression is elevated in isolated sensory
neurons compared with the levels in DRG explant culture, suggesting growth factor receptors are modulated in culture by axotomy56.
However, these conditions can also be exploited to study axonal
regeneration in a model where the extracellular environment can be
modified more directly than in vivo.
The protocols provided here are optimized for mouse DRG and
TG cultures. Although sensory neurons from many different
vertebrate species (e.g., frog, chick, rat, mouse and even human)
have been isolated and cultured, mice have a particular genetic
advantage, given the many publicly available transgenic mouse lines
in which genes have been deleted or altered or misexpressed.
Sensory neurons cultured from genetically modified mice provide
a powerful experimental tool.

. Heparin flush lock (100 U ml1) (Hospira Inc., cat. no. NDC 0409-1152-70)
. 15 mm round coverslips (Warner, cat. no. 640703CS 15R)
. 2,2,2 tribromoethanol (Sigma, T48402)
. t-amyl alcohol (Sigma, 152463)
. HEPES (Sigma, cat. no. H4034)
. L15 culture medium (GIBCO, cat. no. 11415)
. Percoll, sterile (GE Healthcare, cat. no. 17-0891-01)
EQUIPMENT

. Cole Palmer perfusion pump, model 7520 (Chicago, IL)

. Dissection microscope (SZH-10, Olympus)
. 5 ml polypropylene tubes (Falcon 2063 tubes)
. 12-well culture plate (Falcon 3043)
. 1 pair #5 forceps (Fine Science Tools, cat. no. 11253-20)
. 1 pair #3 forceps (Fine Science Tools, cat. no. 11231-30)
. 1 pair #2 forceps (Fine Science Tools, cat. no. 11223-20)
. 1 pair spring scissors (Fine Science Tools, cat. no. 15012-12)
NATURE PROTOCOLS | VOL.2 NO.1 | 2007 | 153

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2007 Nature Publishing Group http://www.nature.com/natureprotocols

. 1 pair student vannas spring scissors (for initial training) (Fine Science
Tools, cat. no. 915009-09)
. 1 pair fine iris scissors (Fine Science Tools, cat. no. 14094-11)
. Hemostat
. 1 cc syringe with 25-gauge needle
. 22-gauge needle
. 30-gauge needle
. 60 cc syringe
REAGENT SETUP
Animals The protocol outlined below is optimized for culture of sensory
neurons from a 68-week-old mouse. Protocols are given for both DRGs and
TGs. m CRITICAL Tissues obtained from embryonic or neonatal animals require
less enzymatic digestion and milder trituration for optimum health. Culture
of sensory neurons from older mice (older than 6 months) or rats requires
more aggressive dissociation. ! CAUTION All animal experiments are to
be performed in accordance with relevant authorities guidelines and
regulations.
Anesthesia Mix 1.0 g 2,2,2 tribromoethanol (powder) in 1.0 ml t-amyl alcohol
(liquid) to make avertin. Shake to dissolve. Aliquot 100 ml and store at

20 1C for up to 6 months. Before perfusion, dilute stock 1:40 (vol/vol) in sterile
saline. Use 25 ml diluted avertin per gram body weight injected i.p. (e.g., 0.88 ml
diluted avertin for a 35 g mouse). Warm the solution in a 37 1C water bath
if necessary to mix avertin thoroughly with saline. Note that many IACUCs
will allow avertin to be used only for nonsurvival procedures such as perfusion.
Avertin is not recommended for survival surgeries.
Laminin solution Dilute 1 mg laminin in 5 ml HBSS without Ca++/Mg++.
Aliquot at 100 ml and freeze at
20 1C for up to 6 months.
Lys solution Dissolve 5 mg poly-D-lysine (lyophilized) in 2.5 ml sterile
dH2O. Aliquot at 100 ml and freeze at
20 1C for up to 6 months.

Penicillin/streptomycin solution Make up at 10,000 U per 10 mg ml1 in

sterile H2O. Filter-sterilize and aliquot (1 ml). Store at
20 1C for up to 1 year.
FCS Heat-inactivate serum at 56 1C for 30 min. Aliquot (10 ml) and store
at
20 1C for up to 1 year.
Saturated NaCO3 solution Prepare saturated solution of NaCO3
in sterile
H2O. Store at room temperature (2125 1C) for up to 1 month.
Culture medium Add 50 ml FCS and 5 ml penicillin/streptomycin into 445 ml
F12. Filter-sterilize and store at 4 1C for up to 1 month. Warm the amount
of medium that is absolutely necessary for feeding or culturing cells; do not
warm the entire bottle. Repeated warming hastens the degradation of L-GIn
and other components of the medium.
1 M HEPES Make up in H2O. Filter-sterilize and store at 4 1C for up to
1 month.
L15 complete medium Add 25 ml FCS, 10 ml 1 M HEPES, 5 ml penicillin/
streptomycin into 470 ml L15. Filter-sterilize and store at 4 1C for up to
1 month.
EQUIPMENT SETUP
Perfusion setup Lay out the following tools for perfusion: hemostat, small
scissors, spring scissors, saw-tooth forceps, 1 cc syringe with 30-gauge needle
(for heparin). Place tools near perfusion pump. If you do not have a perfusion
pump, a 60 cc syringe with a 22-gauge needle can be used.
Dissection setup Lay out the following tools for dissection: #5 forceps, #3
forceps, spring scissors, fine iris scissors. Place tools near to dissecting microscope on a large Petri dish and spray liberally with 70% ethanol. Allow to
dry before use. Removal of sensory ganglia can be done on the laboratory bench
top, although some laboratories perform the dissection inside an open-front
laminar flow hood. m CRITICAL If cells are to be cultured for more than 3 d,
dissection inside a hood is preferable, as this reduces the likelihood of
contamination.

PROCEDURE
Culture medium preparation TIMING 10 min
1| Prepare culture medium as described above and filter-sterilize it with disposable bottle filter (0.45 mm).

2| Remove 25 ml culture medium for each mouse to be dissected and place in sterile 50 ml conical tube in tissue culture
incubator.

Coat coverslips TIMING 15 min

3| Thaw laminin aliquot at 4 1C and keep on ice. Thaw one aliquot per two mice.
! CAUTION Laminin can gel if left at room temperature.
4| Make poly-D-lysine solution. Dilute 100 ml poly-D-lysine stock in 10 ml HBSS without Ca++/Mg++. Keep on ice until use;
may be stored at 4 1C for up to 2 weeks.
5| Sterilize coverslips by dipping in 70% ethanol and flaming with a Bunsen burner in tissue culture hood.
6| Set each coverslip on edge in one well of a 12-well culture plate to dry in hood.
7| When coverslips are dry, place them centered on the bottom of the well. Do not allow the coverslip to touch the sides of
the well, or the laminin will wick off the coverslips.
? TROUBLESHOOTING
8| Dilute 100 ml laminin stock in 1.1 ml poly-D-lysine solution in hood.
9| Mix and pipette 100 ml laminin solution onto the center of each coverslip. Take care that laminin solution remains far away
from the walls of the well to ensure coating of coverslip. Laminin must be pipetted onto coverslips within 10 min of making
Lys/laminin solution to ensure adequate coating, so if many coverslips are to be prepared, coat them in batches.
10| Store 12-well plate at 4 1C for at least 1 h (and up to overnight) before use.
? TROUBLESHOOTING

Prepare enzyme solutions TIMING 30 min

11| In tissue culture hood, add 3 ml saturated NaHCO3 solution and 1 mg L-Cys to 1.5 ml Ca++/Mg++-free HBSS.
12| Add 60 U papain to L-Cys solution and place in 37 1C water bath to dissolve papain (approximately 20 min).
154 | VOL.2 NO.1 | 2007 | NATURE PROTOCOLS

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13| In tissue culture hood, add 12 mg CLS2 and 14 mg dispase type II to 3 ml Ca++/Mg++-free HBSS.
14| Place in 37 1C water bath to dissolve collagenase/dispase (approximately 20 min).
15| Filter-sterilize both enzyme solutions and keep in 37 1C water bath until ready for use.
! CAUTION Do not leave at 37 1C for more than 2 h

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Transcardial perfusionoptional TIMING 10 min

16| We recommend a cold perfusion to reduce protease activity and metabolic toxicity and improve cell survival. Also, the
removal of blood aids in dissection. Regardless of whether perfusion is carried out, subsequent steps are identical, starting
from Step 22.
17| For each mouse to be dissected, prepare a perfusion tube (50 ml HBSS without Ca++/Mg++) and a collection tube for DRGs
(3 ml HBSS without Ca++/Mg++). Place both tubes on ice.
18| Set up perfusion pump with intake tubing in perfusion tube in an ice bucket. Attach a 1 cc syringe with a 22-gauge
needle to the perfusion outflow. Each adult mouse will be perfused with 50 ml ice-cold HBSS without Ca++/Mg++ medium
before dissection.
19| Anesthetize animals using 20 ml g1 avertin i.p. Proceed when mouse does not respond to strong squeezing of hindpaw.
20| Expose the heart and inject 0.2 ml heparin into the left ventricle. Snip a small hole in the right atrium with iris scissors,
insert the perfusion needle into the left ventricle and perfuse with 50 ml ice-cold HBSS over 3 min.
21| To isolate trigeminal neurons, go to Step 39. For DRG neurons, continue with Step 22.

Laminectomy TIMING 5 min

22| Move mouse to the dissection area. Spray back fur liberally with 70% ethanol. Using iris scissors, make a large transverse
cut in middle of back skin. With one hand, pinch skin between thumb and forefinger on the caudal side of the cut and with
the other hand pinch skin on the rostral side of the cut. Pull the skin in opposite directions to remove all back skin and expose
cervical, thoracic and lumbar spinal regions. Exposing the back of the animal in this way prevents hair from contaminating
the dissection (Fig. 1a).
23| Perform a laminectomy to remove the roof of the vertebral canal and expose the spinal cord and DRGs. Using spring
scissors, make an incision in the mid-back region exposing the spinal cord (a shallow cut into the cord is all right) (Fig. 1a).
24| Using the #3 forceps, grasp the dorsal edge of the cut vertebra and overlying muscle. Lift up slightly and slide one tip of
the spring scissors into the vertebral canal at the three oclock position and snip bone and surrounding muscle. The depth of
this cut should be 25 mm. Now repeat at the nine oclock position. Repeat cutting from side to side while lifting the growing
tissue flap (Fig. 1b).
25| Work your way to the caudal end of the mouse. Now repeat the process in the rostral direction (Fig. 1c).

DRG dissection TIMING Removal of all ganglia will take 12 h for novices. Once proficiency is established, ganglia
can be collected within 30 min
26| Grasp the dorsal root (located between the DRGs and spinal cord; see Box 1) with the #5 forceps. Cut the spinal nerve
(immediately distal to the ganglion) with the spring scissors and remove any connective tissue attached to the DRGs. Then
cut the dorsal root and remove the DRGs. To remove lumbar DRGs (Fig. 1e), tug gently on the dorsal root to pull DRGs into

BOX 1 | LOCATION OF DORSAL ROOT GANGLIA

There are a total of 60 DRGs in the mouse. DRGs are located in pairs at cervical, thoracic, lumbar and sacral vertebral levels (at coccygeal
levels, there may be sensory neurons associated with the dorsal roots but they are few in number and difficult to identify). In mouse, there
are 8 pairs of cervical, 13 pairs of thoracic, 5 pairs of lumbar and 4 pairs of sacral DRGs. (Rats have six lumbar segments and, hence, a pair
of L6 sensory ganglia, whereas in C57BL/6 mice these ganglia lie within the sacrum and are considered S1; however, it should also be noted
that this may not be true of all mouse strains.) If specific ganglia are to be dissected and cultured, the following landmarks are useful
for identification: C1 ganglia are located just below the skull and above the first vertebra; T13 ganglia are located just below the last rib
(see Fig. 1).

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the vertebral canal, then clip nerves and connective tissue to remove DRGs. Although removal of the spinal cord improves
access to DRGs, experimenters may find it easier to retrieve ganglia with dorsal roots still attached to the cord. Care should be
taken to minimize the amount of nerve collected with the ganglia. Transfer ganglia to 3 ml HBSS without Ca++/Mg++ in a tube
on ice. It is critical to avoid drying out the DRGs at this stage.
m CRITICAL STEP DRGs are located at the four and eight oclock positions within the vertebral canal between pairs of adjacent
vertebrae (Fig. 1cf). Avoid grasping the DRGs directly with forceps. At cervical and thoracic levels, ganglia can be pulled into
the vertebral canal by pushing the spinal cord to one side (Fig. 1c,f).

2007 Nature Publishing Group http://www.nature.com/natureprotocols

Cell isolation TIMING 30 min

27| Carry out Steps 2838 (except incubations and centrifugations) in a tissue culture hood with standard sterile technique.
! CAUTION Use of various proteases to isolate sensory neurons has been reported in the literature; the combination described
here works well for murine sensory neurons. Although we have in the past used trypsin to isolate rat DRG neurons, we have
found it to be too harsh for reliable culture of mouse sensory neurons.
? TROUBLESHOOTING
28| Move to tissue culture hood. Let DRGs settle to bottom of collection tube; carefully remove 1.5 ml HBSS without disturbing
the ganglia. Add papain solution to tissue/HBSS in collection tube and incubate for 10 min in 37 1C water bath.
29| Spin for 1 min at low speed (less than 200g) to pellet ganglia. Carefully remove papain solution.
30| Add collagenase/dispase solution to tissue in collection tube, flick tube gently to agitate ganglia and incubate for 10 min
in 37 1C water bath.
31| Spin for 1 min at low speed to pellet ganglia. Carefully remove collagenase/dispase solution.
? TROUBLESHOOTING
32| Add 2 ml prewarmed culture medium (F12 with 10% FCS, penicillin/streptomycin) to tissue. At this point, the ganglia
should be mildly digested and will stick together in a loose clump.
33| Let tissue settle to bottom of tube (or spin briefly to pellet tissue).
34| Remove 12-well plate from refrigerator and place it at the back of the tissue culture hood to warm to room temperature
while isolation is completed.
35| Carefully remove medium from tissue. Add 0.5 ml culture medium to tissue in tube.
36| Triturate using a fire-polished glass Pasteur pipette until solution becomes cloudy (approximately ten times). Ganglia
should pass through the pipette with friction initially and progress to passing easily during the trituration. It is critical to avoid
introducing air bubbles at this step.
? TROUBLESHOOTING
37| Rinse coverslips with sterile H2O in wells. Aspirate off water; remove obvious droplets but do not allow to dry completely.
38| Plate cells on laminin/poly-D-lysine-coated coverslips. Add 0.7 ml culture medium to triturated DRGs. Pipette several times
to mix. Then carefully pipette 100 ml cell suspension onto each laminin/poly-D-lysine-coated coverslip (in well). Place plate in
37 1C 5% CO2 incubator.
PAUSE POINT Two hours after plating, flood wells with 1 ml warm (37 1C) growth medium. Replace medium with fresh
(warm) culture medium every third day. If desired, growth factors may be added to culture medium when feeding cells. Adult
DRG neurons will survive without addition of growth factors; neonatal (isolated from neonates under 2 d old) and embryonic
neurons will not57. Commonly used growth factors are NGF (50 ng ml1) and Glial cell line-derived neurotrophic factor
(GDNF; 20 ng ml1). Keep in mind that addition of growth factors will affect gene expression as well as neuron survival.
Mouse trigeminal neuron variation
39| The following protocol is optimized for isolation of sensory neurons from mouse TGs, isolation of rat TG neurons is slightly
different4. Steps 120 are the same as described above for DRG isolation. However, in addition to prewarming 25 ml culture
medium per mouse, also prewarm 25 ml complete L15 medium per mouse in 37 1C 5% CO2 incubator.

Collection of TGs TIMING 10 min

40| To expose the TGs, remove the skin from the skull and use the iris scissors to remove the top of the skull and expose the
brain. At the rostral end of the brain, make a cut between the forebrain and the olfactory bulbs. Using the #3 forceps, gently lift
the front portion of the brain to expose the optic nerves. Cut both optic nerves and continue to lift the brain to expose cranial
156 | VOL.2 NO.1 | 2007 | NATURE PROTOCOLS

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nerve V (the trigeminal nerve). Cut this nerve and remove the entire brain from the skull as shown in Figure 2. The TGs are
readily visible in the base of the cranium. Using the spring scissors, cut the three main branches of the TGs and the connection
to the brain (as indicated in Fig. 2). Grasp the ganglion by the posterior end and lift while clipping the connective tissue to
free the TG from the dura mater. Place both ganglia into a 35 mm dish containing 2 ml HBSS without Ca++/Mg++. Keep the dish
on ice.

Cell isolation TIMING 90 min

41| Steps 4357 take place in the tissue culture hood, except for water bath and centrifuge steps. Chop ganglia into
1012 pieces using spring scissors. Transfer tissue pieces to tube containing 1.5 ml HBSS without Ca++/Mg++.

2007 Nature Publishing Group http://www.nature.com/natureprotocols

42| Add papain solution to tissue/HBSS in tube and incubate for 20 min in 37 1C water bath. Gently flick tube to agitate
at 10 min.
43| Spin for 1 min at low speed (less than 200g) to pellet ganglia. Carefully remove papain solution.
44| Add collagenase/dispase solution to tissue in hood, flick tube gently to agitate ganglia and incubate for 20 min in 37 1C
water bath. Gently flick tube to agitate at 10 min.
45| Spin for 4 min at 400g to pellet tissue. Carefully remove collagenase/dispase solution.
46| Add 0.5 ml prewarmed complete L15 medium (L15 with 5% FCS, penicillin/streptomycin, HEPES) to tissue. At this point,
the ganglia should be greatly digested and will stick together in a loose clump.
47| Triturate by passing tissue through a 200 ml pipette tip 23 times. Solution will become very cloudy, although clumps of
nerve will still be visible. It is critical to avoid introducing air at this step.
48| Make Percoll gradient to separate myelin and nerve debris from trigeminal neurons. In tissue culture hood, add 1.1 ml
Percoll to 2.9 ml warm complete L15 in 15 ml tube (28% Percoll). In another tube, add 0.5 ml Percoll to 3.5 ml warm complete
L15 (12.5% Percoll). Gently layer the 12.5% Percoll over the 28% Percoll solution.
49| Gently layer the 0.5 ml cell suspension over the Percoll gradient. Do not disturb the lower Percoll layers. Spin for 10 min
at 1,300g.
50| Remove 12-well plate from refrigerator and place it at the back of the hood to warm to room temperature while isolation is
completed.
51| Cells will pellet to bottom of tube; debris will be easily discernable at Percoll interface.
52| Remove (and discard) top 4.5 ml including interface with debris.
53| Add 4 ml complete L15 to remaining solution in tube. Spin for 6 min at 1,000g.
54| Cell pellet will be visible at bottom of tube. Remove medium from tube and resuspend pellet in 600 ml complete F12.
55| Rinse coverslips with sterile H2O in wells. Aspirate off water; remove obvious droplets but do not allow to dry completely.
56| Plate cells on laminin/poly-D-lysine-coated coverslips. Then carefully pipette 100 ml cell suspension onto each laminin/
poly-D-lysine-coated coverslip. Place plate in incubator.
PAUSE POINT Two hours after plating, flood wells with 1 ml warm (37 1C) growth medium. Replace medium with fresh
(warm) culture medium every third day. If desired, growth factors may be added to culture medium when feeding cells.

TIMING
Steps 12: 10 min (expert); 15 min (beginner)
Steps 310: 15 min (expert); 40 min (beginner)
Steps 1115: 30 min (expert); 40 min (beginner)
Steps 1626: 45 min (expert); 75 min (beginner)
Steps 2738: 30 min (expert); 60 min (beginner)
Step 40: 10 min (expert); 20 min (beginner)
Steps 4156: 1.5 h (expert); 3 h (beginner)
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
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2007 Nature Publishing Group http://www.nature.com/natureprotocols

TABLE 1 | Troubleshooting table.

Step
310

Problem
Very few cells adhere; adherent
cells do not extend processes

Possible cause
Dishes or coverslips are not well
coated with laminin

Solution
Verify that laminin was kept on ice before coating and
that laminin did not wick off coverslip during coating;
check expiration date on laminin/laminin stock; check
expiration date on Lys; make new Lys working solution
(in HBSS); order new lot of laminin

67

Laminin wicks off coverslip during

coating

Coverslips were not completely dry

before laminin was added

Dry coverslips overnight in hood (in 12-well plate)

before coating

2227

Cells become contaminated soon

after plating

Contamination introduced during

dissection

Spray tools liberally with 70% ethanol before dissection; use separate tools for perfusion and dissection;
change gloves after dissection/before culture; dissect
in hood

2831

Trituration is difficult (tissue does

not easily separate)

Tissue was not well digested

Verify that the temperature of water bath is 37 1C;

verify that enzymes are not left in water bath for more
than 1 h before use; check expiration dates on
enzymes; order new lot of enzymes

36

Low cell yield; excessive debris in

culture

Over-trituration; too much air

in trituration

Triturate with larger-bore pipette; triturate fewer

times; take more care to reduce bubbles in trituration
process

36

Low cell yield; chunks of tissue

remain intact

Under-trituration

Triturate with smaller bore pipette; increase number of

triturations

2836

Cells do not appear healthy after

plating

Over-trituration; over-digestion;
wrong pH of medium; wrong
temperature/atmosphere of
incubator

Reduce trituration number; reduce digestion time;

check pH of medium (adjust to 7.4); check incubator
temperature (should be 37 1C) and CO2 pressure (should
be 5%)

ANTICIPATED RESULTS
A DRG dissection yielding 40 ganglia can be plated onto 12 coverslips (15 mm) at approximately 5  104 cells/coverslip.
A TG dissection of two ganglia can be plated onto six coverslips (15 mm) at approximately 5  104 cells per coverslip.
Evaluating the health of cultured neurons
Neuronal cell bodies should appear round, smooth and phase-bright at plating. Most of the cells should attach well to a
laminin-coated substrate within 2 h of plating, and axonal processes should be easily discernable 24 h after cell isolation.
Examples of healthy DRG cultures following 2 h, 1 d and 3 d in vitro are shown in Figure 3. A functional standard for cell health

Figure 3 | Isolated DRG neurons visualized with an antibody to the

high-molecular weight neurofilament protein, which preferentially labels
neurons with higher-caliber axons. Neurons rapidly develop profuse axonal
arborizations on the laminin substrate. (a,b) Adherent neurons on the day
of plating, after medium has been added to the coverslips. Axon stumps
are visible. (c,d) By 24 h after plating, many neurons have begun to
extend neurites. (e,f) By 3 d after plating, an extensive net of axons is
present. a, c, e, 100 original magnification; b, d, f, 200 original
magnification; scale bars 250 mm.
158 | VOL.2 NO.1 | 2007 | NATURE PROTOCOLS

PROTOCOL

2007 Nature Publishing Group http://www.nature.com/natureprotocols

is equally important. Neurons should respond vigorously to acute depolarization, evaluated using electrophysiology or calcium
imaging after cells attach to coverslip (as soon as 2 h after plating). Healthy isolated neurons will express ion channels,
receptors, neuropeptides, calcium-binding proteins and/or cytoskeletal components detected in vivo; these proteins may be
used as immunohistochemical markers in vitro. If cells are to be cultured for extended periods (more than 3 d), it is important
to remember that non-neuronal cells will proliferate during this time in culture, consuming more nutrients and releasing more
endocrine factors than at day 1. A variety of techniques have been developed to minimize the presence of non-neuronal cells in
culture. These include preplating cell lysates to remove non-neuronal cells before culture, use of DNA topoisomerase inhibitors58
and the use of mitotic inhibitors such as Floxuridine (FUdR) and uridine; cytosine arabinoside is not recommended27,59.
How well does the neuronal population in culture represent the diversity of DRG cell types present in vivo?
Sensory neuron culture reproduces the broad diversity of neuronal cell types and response properties seen in vivo. However, it is
important to remember that response properties change over time in culture in response to both the dissociation procedure and
the altered environment. Furthermore, there is extensive evidence that growth factors play a pivotal role in the maintenance of
neuronal phenotype and excitability, and all sensory neurons express growth factor receptors of some type. For example, NGF has
recently been shown to regulate expression of TRPV160, a heat-sensitive channel; TRPM8, a cold-sensitive channel61; and ASIC3,
an acid-sensitive channel62. Thus NGF, in combination with other factors, may modulate the heat, cold and/or acid sensitivity of
some sensory neurons. Also, NGF, artemin, GDNF and neurturin have all been shown to acutely sensitize heat responses in vivo
and TRPV1 channel activity in vitro63,64. Therefore, culture conditions must be carefully tailored to experimental design.

ACKNOWLEDGMENTS This research was supported by National Institute of

Neurological Disorders and Stroke Grants NS 311826 (B.M.D.) and National
Institute of Diabetes and Digestive and Kidney Diseases Grant DK 063922 (S.A.M.).
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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