VERSION 1.1.0
Table of Contents
1.0 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Storage of Medium and Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3 Additional Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
1
1
2
2
2
4
4
4
6
6
6
7
Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Blocking and Labeling with Primary Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Secondary Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
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1.0 Materials
1.1 Storage of Medium and Supplements
Very important! Upon arrival, cryopreserved neurospheres* must be stored IMMEDIATELY at -135C or colder, or in liquid nitrogen.
NeuroCult NS-A Basal Medium (Rat)* should be stored at 2 - 8C. NeuroCult NS-A Proliferation Supplements (Rat)* and NeuroCult
NS-A Differentiation Supplements (Rat)* should be stored at -20C.
After NeuroCult NS-A Proliferation Supplements (Rat) have been added to the NeuroCult NS-A Basal Medium (Rat), storage at
2 - 8C is recommended for no more than 1 month. Long-term storage at 2 - 8C is not recommended. Refer to Section 2.0 for
preparation of Complete NeuroCult NS-A Proliferation Medium (Rat).
After NeuroCult NS-A Differentiation Supplements (Rat) have been added to the NeuroCult NS-A Basal Medium (Rat) to make "Complete"
NeuroCult NS-A Differentiation Medium (Rat), storage at 2 - 8C is recommended for no more than 1 month. Long-term storage of the
"Complete NeuroCult NS-A Differentiation Medium (Rat) at 2 - 8C is not recommended.
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Component
Final Concentration
10 g/mL rh EGF
2 L
20 ng/mL
10 g/mL rh FGF-b
1 L
10 ng/mL
0.2% Heparin
1 L
0.0002% (2 g/mL)
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3.2 Preparation of Cells from Cryopreserved Neurospheres
3.2.1 Thawing Cryopreserved Rat Neurospheres
1. Warm Complete NeuroCult Proliferation Medium (Rat; made up as in Section 2.0) to 37C.
2. Add 9 mL of Complete NeuroCult Proliferation Medium (Rat) to a sterile 14 mL tube.
3. Remove the cryovial containing the cryopreserved E18 neurospheres from rat cortex (Catalog #00340) from the freezer and
thaw quickly in a 37C water bath.
4. Using a 1 mL disposable tip attached to a P1000 micropipettor, add 1 mL of Complete NeuroCult Proliferation Medium (Rat)
dropwise to the cryovial containing thawed neurospheres. Transfer the cell suspension to the tube prepared in step 2 and
centrifuge at 90 x g (~400 rpm) for 5 minutes.
5. Aspirate off all the supernatant. Add 10 mL of Complete NeuroCult Proliferation Medium (Rat) to the pellet and resuspend
neurospheres by pipetting gently (DO NOT DISSOCIATE NEUROSPHERES).
6. Transfer the entire cell suspension to a T-25 cm2 flask (Nunc Catalog #156367 or VWR Catalog #15708-130).
7. Observe the cell suspension under an inverted light microscope to determine appearance of neurospheres. The morphology of
the neurospheres may not look like intact spheres at this point due to the freeze/thaw process (the morphology should
recover the following day). This day (the day the neurospheres are thawed) is considered day 1. Culture in a humidified
incubator at 37C and 5% CO2.
8. Check cultures the next day and observe the morphology of the neurospheres. The neurospheres should mostly be floating
single neurospheres which look intact and viable (spheroid appearance) with only a minority of the neurospheres (<20%)
stuck down to the bottom of the flask. The neurospheres should be passaged at this point using the procedure described in
Section 3.2.2.
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5.3 Differentiation Assay
1. If using the BD BioCoat 8-well culture slide (pre-coated with Poly-D-Lysine/Laminin BD Catalog #354688, or Poly-D-Lysine
Catalog #354632) add 0.75 mL/well of "Complete" NeuroCult NS-A Differentiation Medium (Rat). If using Poly-L-Ornithine
coated glass coverslips prepared as described in Section 5.2.1, place a single prepared coated coverslip into an individual
well of a 24-well culture dish (e.g. Corning Catalog #3526) containing 1 mL/well of "Complete" NeuroCult NS-A
Differentiation Medium (Rat).
2. After 3 - 4 days of culturing rat neurospheres in "Complete" NeuroCult NS-A Proliferation Medium (Rat), remove the medium
with suspended cells and place in an appropriate sized sterile tissue culture tube. Spin at 90 x g (~400 rpm) for 5 minutes.
3. Remove all of the supernatant and discard.
4. Resuspend the neurospheres in 150 - 180 L "Complete" NeuroCult NS-A Differentiation Medium (Rat). With a P200 pipette
tip, triturate the neurosphere suspension until single cell suspension is achieved (refer to Section 4, Steps 2 and 3 for a
description of trituration).
5. Resuspend the single cell suspension with 10 mL of Complete NeuroCult NS-A Differentiation Medium (Rat). Spin at
150 x g (~800 rpm) for 5 minutes. Remove all of the supernatant and discard.
6. Resuspend the cell pellet in approximately 200 L of Complete NeuroCult NS-A Differentiation Medium (Rat).
7. Measure the precise volume and count cell numbers using a dilution in Trypan Blue (1/5 or 1/10 dilution) and hemacytometer.
8. Prepare cells in an appropriate volume of Complete NeuroCult NS-A Differentiation Medium (Rat). Cells should be plated
with a density of 5 x 105 cells/mL on a coated-coverslip in a 24-well plate or at 1 x 105 cells/well in a BioCoat 8-well
chamber slide.
9. Observe cultures after 5 - 10 days with an inverted light microscope and determine if cells have differentiated and are viable.
10.Plates should be checked routinely to determine if the medium needs to be changed during the differentiation procedure. If
the medium becomes acidic (yellow), a half-medium change should be performed by removing approximately 50% of the
medium and replacing with fresh "Complete" NeuroCult NS-A Differentiation Medium (Rat).
11.Coverslips or pre-coated chamber slides containing differentiated rat neural cells can be removed after 5 - 10 days and
processed immediately for indirect immunofluorescence as described in Section 6.0.
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6.2 Permeabilization
1. Permeabilize cells by adding 1 mL of PBS containing 0.3% Triton X-100 (Sigma Catalog #T9284) to each well and incubate for
5 - 10 minutes at room temperature.
2. Remove PBS/Triton-X 100 by aspiration. Perform 2 x 5 minute PBS washes.
Secondary Antibody
Serum
Catalog #
goat
goat
goat
10211
10212
10213
goat
10214
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2. Dilute primary antibody in the appropriate serum-containing diluent (from Table 1). Appropriate working dilutions for
immunolabeling are shown in Table 2 (below).
Table 2: The optimal working dilution used for different primary antibodies
Targeted Antigen
Clone
Isotype
Catalog #
Working Dilution
GABA
rabbit polyclonal
01411
1:200
rabbit polyclonal
01415
1:100
AP20
mouse IgG1
01410
1:200
rabbit polyclonal
01417
1:200
Nestin
Rat 401
mouse IgG1
01418
1:50
TUJ1
mouse IgG2a
01409
1:1000
Oligodendrocyte Marker O4
O4
mouse IgM
01416
1:50
RIP
mouse IgG1
01433
1:100
Tyrosine Hydroxylase-2
TH2
mouse IgG1
01412
1:400
The working dilutions are only recommendations as the optimal working dilution for each specific application should be
determined by the user.
3. Add diluted primary antibodies to the 24-well plate or chamber slides in a minimum volume of 250 L. Alternately place a
small volume of antibody, approximately 50 L, directly on the coverslip containing the differentiated cells and place a clean
second coverslip directly on top. Place in a hydrating chamber.
4. Incubate for 2 hours at 37C or overnight at 2 - 8C.
5. Wash off primary antibody with 3 x 5 minute washes using PBS.
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6.5 Mounting
If pre-coated chamber slides are used, perform Steps 1 - 2. If glass coverslips are used, then follow Steps 3 - 4.
1. If pre-coated 8-well chamber slides are used, follow manufacturer's protocol for removal of the chambers from the glass
slides. Rinse slides in distilled water in a coplin jar.
2. Add about 5 L of mounting medium (e.g. FluorSave Reagent, Calbiochem Catalog #345789) in each chamber slot and cover
with a 75 mm coverslip avoiding trapping any air bubbles.
3. If coverslips are used, add 10 L of mounting medium (e.g. FluorSave Reagent, Calbiochem Catalog #345789) to a clean
glass coverslip. Remove immunostained coverslip from the 24-well plate and gently tap corner of the coverslip to remove
excess water.
4. Place coverslip cell side down onto the mounting medium avoiding any air bubbles.
5. Visualize immunostaining under a fluorescent microscope using the appropriate filters for each fluorochrome (Table 3). Refer
to Section 8.0 for representative photographs of immunolabeled differentiated rat neurospheres.
Table 3: Peak wavelengths of absorption and emission for different fluorochrome-conjugated secondary antibodies
Fluorochrome
Aminomethylcoumarin, AMCA
Fluorescein Isothiocyanate, FITC
Rhodamine Red-X, RRX
350
492
570
450
520
590
Texas Red, TR
596
620
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Figure 7. Differentiated rat neural cells cultured with NeuroCult NS-A Differentiation Medium (Rat). Cells are immunostained with lineage
specific antibodies as described in Section 6.3 and counterstained with DAPI (blue).
A. -Tubulin (red) and MBP (Green)
B. Oligodendrocyte Marker O4 (green) and -Tubulin (red)
C. Nestin (red)
D. GFAP (green) and MAP2 (red)
E and F. Triple staining with Oligodendrocyte Marker O4 (green), GFAP (blue) and -Tubulin (red)
Magnification: 400X
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