Henderson Lab Protocol
http://blogs.cuit.columbia.edu/hendersonlab/
Dr. Christopher Henderson, Ph.D.
Principal Investigator
ch2331@columbia.edu
Dominick Papandrea
Lab Manager
dp2559@columbia.edu
Mouse motor neuron primary culture
Materials and Methods
Embryonic age
The window for the correct age of mouse embryos to be dissected is much smaller than that for rat
or chicken embryos. We use E 12.5 mouse embryos, but since it is sometimes difficult to obtain the
exact age, we dissect our embryos in the late morning or early afternoon. Embryos younger than this
age are extremely difficult to dissect, specially when pulling the meninges out from the spinal cord.
When we have used older embryos, such as E 13 we obtain approximately half the motoneurons than
the number obtained at E 12.5. The number of motoneurons obtained with E 14 embryos is
negligible, maybe because of the difficulties in dissociating older spinal cords.
The way we calculate the gestation date of embryos is the following: Light is switched on at 6:30
am and switched off at 18:30. Female and male rats are put together for mating at 17:30. They are
then separated next day at 8:00 am which will is considered as day zero after detection of the vaginal
plug, and the development of the embryos is timed from this date.
Experimental methods
Dissection of spinal cords
The dissection procedure (described below) of embryonic spinal cords is the same as that described
for rat embryonic spinal cords (Henderson et al.., 1995).
Center for Motor Neuron Biology and Disease, Columbia University Medical Center, Physicians and Surgeons Building, Room 5-420, 630 West
168th Street, New York, NY 10032. Tel: +1 212-342-4086 Fax: +1-212-342-4512 E-mail: ch2331@columbia.edu
Equipment and reagents
All dissection instruments may be obtained from Fine Science Tools.
- Scissors (Moria MC 22 No. 14370-22), forceps, and a small perforated spoon (Moria MC 17, No.
10370-17), for embryo removal and handling.
- A silicone base for dissection, made by polymerizing RTV 141A/B (Rhône-Poulenc or similar) in
the bottom of Pyrex Petri dishes with the incorporation of enough activated charcoal to give a black
colour (dishes can be autoclaved).
- Two pairs of fine forceps (Moria MC 17 No. 11370-40), one scalpel No. 15 blade for dissection
and one pair of very fine forceps (Dumont No. 55 forceps, Biologie tip, No. 11255-20) for pulling
out the meninges.
- PBS CA/Mg free.
- A stereoscopic dissecting microscope.
Method
1. Remove membranes surrounding embryos and store at room temperature in CMF-PBS for as short
time as possible. If genotype analysis is performed, embryos should be kept at 4 oC in Hybernate E
medium supplemented with B 27 (Table 1) until dissection is performed. Dissection should be done
in this medium.
2. Transfer to a silicone support under a dissecting microscope. Remove head, tail and bulk of viscera
using forceps as scissors.
3. Use one pair of closed forceps to stabilize the embryo in the dish. Insert one point of the other pair
into the central canal of the rostral spinal cord. Close forceps and use them as scissors to tear dorsal
tissue away over a distance of about 2 mm. Repeat this operation so as to open the spinal cord
dorsally over the whole rostro-caudal extent of the embryo .
4. Insert one pair of closed forceps between the dorsal lip of the spinal cord and and the surrrounding
tissue at a point approximately opposite the forelimb bud. Slide the forceps rostrally and then
caudally along the line of lowest resistance. Turn the embryo and repeat the operation on the other
side; the cord should be well separated, although some meninges and dorsal root ganglia remain
attached.
5. After freeing the ventral face of the spinal cord from the embryo, remove the whole cord. Hold
the rostral end of the cord with forceps and pull the meninges away in a rostro-caudal direction.
6. Remove the dorsal half of the spinal cord: lay the cord flat on a silicone support with its ventral
face up, and cut along the middle of each side using a scalpel.
Preparation of suspension of dissociated spinal cord cells
For the majority of experiments we use 6 embryos, which gives a yield of 60,000-90,000
motoneurons. The following procedure is intended for 6 spinal cords per centrifugation tube up to
the step of the centrifugation through a BSA cushion. During the Optiprep centrifugation, the
equivalent of 3 spinal cords per tube are used. In the following centrifugation through a BSA cushion,
3 spinal cords per tube are used. For the magnetic cell sorting with indirect microbeads, the
dissociated cells from all spinal cords are pooled together in a single tube.
1. Cut each cord into about 12 pieces using a scalpel.
2. Transfer the fragments of the equivalent of 6 spinal cords to a 15 ml polystyrene tube in 1 ml
modified Ham 10 medium (without L-glutamine, calcium, magnesium, phenol red; Gibco special
preparation, catalogue number: 041-91708 M).
3. Add 8 l trysin (2.5 % w/v; final concentration 0.025%). Incubate for 8 min at 37o C.
4. During the incubation with trypsin prepare each 15 ml polystyrene tube with the following:
complete L 15 medium -bicarbonate
BSA (4% w/v)
Dnase (1 mg/ml in L 15 medium)
0.8 ml
0.1 ml
0.1 ml
5. Immediately after the incubation with trypsin, transfer the fragments of the spinal cords unto the
tubes with the ingredients described in step no. 4.
6. Agitate vigorously by hand until the tissue fragments are disaggregated. Triturate two times gently
with a Gilson blue tip. Allow the fragments to settle during 2 min. Collect the supernatant from each
tube and transfer to another 15 ml polystyrene tube.
7. To each tube tube containing the tissue fragments add:
complete L 15 medium -bicarbonate
BSA (4% w/v)
Dnase (1 mg/ml in L 15 medium)
0.9 ml
0.1 ml
20 l
Triturate six times with a Gilson blue tip. Allow the fragments to settle during 2 min. Collect the
supernatants from each tube and transfer to the 15 ml polystyrene tubes used in step 6 containing the
supernantants.
8. If there are still tissue fragments visible repeat the procedure described in step 7, only that this
time triturate 10 times with a Gilson blue tip.
9. Prepare a 1.5 - 2 ml BSA (4% w/v) cushion for each tube containing the pooled supernatants using
a long Pasteur pipette and gently disposing the BSA solution onto the bottom of each tube. Centrifuge
for 5 min at 470 g.
10. Remove most of the supernatant and resuspend gently 6 times with a Gilson blue tip with 1 ml
of complete medium -bicarbonate. Pool the supernatants and count in a counting cell under phasecontrast. Cells should be completely disscociated. Expect 1-2 x 106 cells/spinal cord.
Optiprep density centrifugation
1. Volumetrically prepare 5.2 % (v/v) solution of Optiprep (Nycomed Pharma) in 4.4% glucose in
Tricine Buffer 10mM, pH 7.8. Sterile filter (0.22 m). For current use keep the buffer at 4 oC and
prepare the mixture with Optiprep just before use. Filter the solution before use.
2. Add 1 ml of complete L 15 medium without bicarbonate to each tube with the dissociated spinal
cord cells. Resuspend 4 times and transfer 1 ml from each tube onto a new polystyrene tube (if you
started with six embryos you will have at this stage 2 tubes, that is the equivalent of 3 spinal cords
per tube. Add 5 ml of L15* (*= buffered with CO2) medium, put 1.5 - 2 ml Optiprep solution under
each tube using a long Pasteur pipette by gently disposing the solution onto the bottom of each tube.
The interface should be sharp.
3. Centrifuge for 15 min at 830 g in a bench top centrifuge at room temperature. Reduce vibration
by switching off the brake. There should be a pellet at the bottom of the tube (small cells) and a
turbid band at the medium-Optiprep interface (large cells).
4. Using a Gilson blue tip, collect the band in 1 ml of medium, including some Optiprep at the
interface. Pool the collected bands from two tubes onto one tube (at this stage you should have 2
tubes). Dilute up to 10 ml with L 15* ( medium to lower the density. Label the tubes to mark the
location of the pellet and collect the cells by centrifugation through a BSA cushion. Remove all of
the supernatant by aspirating carefully with a Pasteur pipette together with a yellow tip. Either count
the cells and put in culture (about 70% motoneurons) or continue with protocol below (about 95%
motoneurons). The following protocol works well only if you can find a nice p75 antibody.
Magnetic cell sorting with indirect microbeads
1. Prepare 40 ml of L15* /BSA 0.05%. Avoid bubbles as this can clog up the column.
2. Prepare a solution of 50 l of L15*/BSA and 50 l anti mouse p75 monoclonal antibody
(supernatant of hybridoma) and resuspend the cells from both tubes in a total volume of 100 l using
a blue tip (6 times). Take 2 l from this cell suspension and dilute up to 20 l with L15*/BSA and
count in a counting-cell under phase-contrast. Expect approximately 100,000 (predominantly large)
cells per cord.
3. Incubate 30 minutes in the fridge (6-12oC).
4. Wash the cells with 10 ml L15*/BSA by pipetting 6 times up and down.
5. Centrifuge through a 4% BSA cushion (12-18oC) and aspirate all of the supernatant. Resuspend
the cells in 80 l L15*/BSA + 20 l goat anti rat IgG microbeads (Quantum magnetics microbeads
250nM), 6 times with a blue tip.
6. Incubate 15 minutes in the fridge (6-12oC).
7. Wash the cells with 10 ml L15*/BSA by pipetting 6 times up and down.
8. Centrifuge through a 4% BSA cushion (12-18oC) and aspirate all of the supernatant. Resuspend
the cells in 500 l L15*/BSA. Alternatively, you can resuspend the cells in 10 ml complete L 15
medium -bicarbonate and stop for one hour at this point. After this, centrifuge through a BSA
cushion, remove the supernatant completely and resuspend in 500 l L15*/BSA.
9. Attach magnet to the stand. Place the separation column in the mini Macs magnet.
10. Attach the flow resistor (23 G needle) to the column.
11. Fill the column by pipetting 1 ml L15*/BSA on top of the column. Let the buffer flow through
and discard the effluent.
12. Apply the magnetically labelled cells (500 l) onto the column.
13. Let the negative cells pass through and collect the effluent as a negative fraction on a centrifuge
tube.
14. Rinse the column 3 times with 500 l L15*/BSA. Always let the entire amount of buffer flow
through the column before you apply new buffer onto the column. Collect in the tube containing the
negative fraction.
15. Remove the column from the magnet and the flow resistor from the column and place the column
on another centrifuge tube.
16. Apply 1 ml of L15*/BSA onto the column and collect the positive fraction. Apply 500 l of
L15*/BSA and flush out the solution with gentle pressure using the plunger supplied with the
column.
17. Centrifuge through a 4% BSA cushion and resuspend the cells in complete neurobasal medium.
Observe under phase-contrast. Expect between 10,000 to 15,000 cells per embryo.
Protocol 4. Culture of motoneurons
1. Purified motoneurons may be used immediately or stored for several hous in the refrigerator
in 5 ml of complete neurobasal medium (Table 6).
2.
Coat tissue culture dishes (Nunc 4 well dishes, ref. no. 176740A) with polyornithin-laminin
(stocks solutions in Table 1). Incubate polyornithin (diluted 1 :1000 in double distilled water)
at room temperature for 30 minutes to overnight. Remove solution and allow to dry in the
hood. Add laminin (500 l for 4 well and 2 ml for 35 mm dishes respectively) diluted 1 :500
in L 15 + bicarbonate tTable 4). Incubate for 2 h to overnight at 37oC in the CO2 incubator.
3. Remove laminin and replace immediately with complete neurobasal medium. Suitable growth
promoting supplements depend on the experimental design. For optimal growth conditions
(such as those required for immunofluorescence) a cocktail of factors including 100 pg/ml
GDNF, 500 pg/ml CNTF may be used.
4. Add motoneurons at a density appropriate for the experiment. For studies of cell survival use
800 motoneurons For immunofluorescence, use 5000 cells per coverslip per well of 4 well
dishes (or 4 well labteks). The coverslips are previously sterilized under U.V. using the
sterilization program of the Strata Linker and then washed in L 15 + bicarbonate (Table 4) for
3 hours minimun to overnight. The contents of each well are aspirated once into a blue
micropipette tip and then gently expelled in order to ensure uniform distribution of
motoneurons. To evaluate motoneuron survival, wells (from 4 well dishes) are filled up to the
brim with warm L 15 medium, and the cover of the dish replaced in order to provide good
phase cpnrast optics over the whole surface.
5. `Nerurite outgrowth should be extensive after the first night in culture. The purity of the
culture may be confirmed by using antibodies against islet-1 (Developmental Hybridoma
Bank).
________________________________________________________________
Table 1. Reagents requiered for motoneuron purification
________________________________________________________________
Product
Source
Solution
Aliquots
Store
Comments
BSA
Sigma
4% (w/v)
10 or 50 ml
-20 oC
Dialyse against L-15a
You can also use the tissue culture BSA solution 7.5%
Dnase I
A 9418
in L-15
Sigma
DN-25
1 mg/ml
in L-15
F 10 mediumb Gibco-BRL
____
modified
041-91708
-20 oC
500 microl
500 ml
4 oC
------
5 or 20 ml
-20 oC
____
1 ml
4 oC
Look at the expiry date
Sigma H 1138 or
Gibco-BRL 26050-039
10 ml
-20 oC
Tests batches before
ordering
Hybernate E
medium
Gibco-BRL
100741-015
100 ml
4 oC
L-15 medium
Gibco-BRL
11415-049
____
500 ml
4 oC
Laminin
ice to
Becton
1.5 mg/ml
500 microl
Glucose
any
Goat anti-rat
IgG microbeads
Miltenyi
Biotec
485-02
Horse serum
72 mg/ml
in L-15
____c
-70 oC
Dickinson
in PBS (500 x)
40232 or home made
Metrizamide
Serva
6.5 % in L 15
Always thaw on
prevent gelling, then keep
at 4 oC
10 ml
-20 oC
For current use, keep at
4 oC
Neurotrophins Genentech
10 microg/ml in
Regeneron
PBS + 10 %
50 microl
Sigma
horse serum
R& D Systems
-70 oC
Stable for 3-4 weeks at
4 oC
PBS/BSA
PBS:any
4 %BSA:
as above
0.5 % BSA
40 ml
(8 fold dilution
of 4 % BSA)
in PBS
4 oC
Use one aliquot per
preparation as it gets
easily contaminated.
Avoid bubbles
Penicillinstreptomycin
Gibco-BRL
15070-022
5000 IU/ml
5 ml
5000 microg/ml
--20 oC
____
Poly-D,LSigma
ornithine
P8638
distilled water
3 mg/ml
in water
50 microl
rat anti mouse Chemicon
1 mg/ml
----
--20 oC
2-8oC
Use double
Stable for 6 months
p 75 antibody MAB357
Sodium
bicarbonate
Gibco-BRL
25080-060
7.5 % (w/v)
in water
100 ml
RT
____
Gibco-BRL
2.5% (w/v)
50 microl
--20 oC
____
25090-010
______________________________________________________________________________________
___
a. The BSA solution is first dialysed against PBS or HBSS using Spectra/Por membranes (MWCO: 25,000,
reference: 132 127, from Spectrum) overnight. Rinse the membranes thoroughly with distilled water before
use. Then, dialyse against L-15 medium for 2-3 days. Filter the solution and aliquot. Freeze if not used
immediately. For current use, it may be stored at 4 oC for 1-2 months. One aliquot of 10 ml should be used
per preparation since it gets contaminated very easily.
b. Costum made media: without L-glutamine, Ca2 and Mg2 and phenol red.
c. Concentration not given by the manufacturers.
Trypsin
______________________________________________________________________________________
___
Table 2. Equipment
______________________________________________________________________________________
___
Producta
Reference
Mini Macs magnet
421-02
Macs multistand
423-03
Large cell separation columns
422-02
______________________________________________________________________________________
___
a. All products from Miltenyi Biotec
________________________________________________________________
Table 3. Modified N2 supplement for culture mediuma
________________________________________________________________
Supplement
Stock concentration
Recipe
Insulin
I6634
500 microg/ml (100 x)
Dissolve 2.5 mg in 0.5 ml 0.1 M HCL; add 4.5
ml water
Putrescine
P 5780
10-2M (100 x)
Dissolve 8 mg in 5 ml PBS
Conalbumin
C 7786
10 mg/ml (100 x)
Dissolve 50 mg in 5 ml PBS
Sodium selenite
S 5261
3 x 10-5 M (1000 x)
Dissolve 1 mg in 19.3 ml water, adjust pH to 7.4,
then dilute tenfold further
Progesterone
2 x 10-5 M (1000 x)
Dissolve 1 mg in 1.6 ml 80 % ethanol; dilute 100P 9783
fold further in ethanol
________________________________________________________________
a. All reagents from Sigma. Store tubes with ‘IPCS mix’ (500 microl insulin, 500 microl putrescine, 500
microl conalbumin, and 50 microl selenite) at --20 oC. Store progesterone separately at --20 oC.
___________________________________
Table 4. Composition of L 15 medium -bicarbonate
___________________________________
L-15 medium
88.8 ml
Glucose
5 ml
Penicillin-streptomycin
1 ml
Progesterone
0.1 ml
IPCS mix
3.1 ml
Horse serum
2 ml
___________________________________
After preparation correct pH to red-orange colour using dry ice,
o
C.
‘L 15 + bicarbonate’ is L 15 alone with the indicated concentration
of bicarbonate.
________________________________________________________________
Table 5. Reagents required for 'complete neurobasal medium'a
________________________________________________________________
Product
Reference
Solution
Aliquots
Store
Comments
Neurobasal medium
21103-049
-----
100 ml
4 oC
expiry date
important
B 27 supplement
17504-036
-----
500 µl
--20 oC
unstable at 4 oC.
Expiry date and
lot number
important
Glutamine
25030
500 µl
--20 oC
keep at 4 oC no
longer than 15
days
Glutamateb
11048
25 mM in
500 µl
L 15
(1000 x stock)
--20 oC
_____
2-mercaptoethanol
21985
25 mM in
500 µl
L 15
(1000 x stock)
--20 oC
expiry date
important
Horse serum
heat inactived
26050-039
--20 oC
keep at 4 oC no
longer than 1
month
-----
10 ml
a All reagents from Gibco BRL
b For long term experiments, glutamate is omitted for medium changes every four of five days
________________________________________________________________
__________________________________________________
Table 6. Composition of 'complete neurobasal medium'
__________________________________________________
Neurobasal medium
95.55 ml
B 27 supplement
2 ml
Glutamine
250 µl
Glutamate
100 µl
2-mercaptoethanol
100 µl
horse serum
2 ml
__________________________________________________