28725 neuro rat pdf

T E C H N I C A L M A N U A LV E R S I O N 1 . 1 . 0

In Vitro Proliferation and Differentiationof Rat Neural Stem And Progenitor Cells (Neurospheres)

Using NeuroCult®

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For Research Use OnlyStemCell Technologies

Version 1.1.0May 2007

Catalog #28725

In North AmericaTel: 604.877.0713Fax: 604.877.0704Toll Free Tel: 1.800.667.0322Toll Free Fax: 1.800.567.2899E-mail: [email protected]

In the United KingdomTel: +44.(0).20.7691.3561Fax: +33.(0).4.76.18.99.63Toll Free within United Kingdom:Tel: 0800.731.27.14Fax: 0800.731.27.13E-mail: [email protected]

In EuropeTel: +33.(0).4.76.04.75.30Fax: +33.(0).4.76.18.99.63e-mail: [email protected]

Table of Contents

1.0 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Storage of Medium and Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Additional Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2.0 Preparation of “Complete” NeuroCult® NS-A Proliferation Medium (Rat) . . . . . . . . . . . . . . 2

2.1 Thawing of Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Preparation of Medium, Supplements and Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.2.1 Preparation Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3.0 Preparation of E18 Rat CNS Cells for In Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1 Preparation of Primary Rat CNS Tissue and Initial Culture of Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . 3

3.2 Preparation of Rat Cells from Cryopreserved Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2.1 Thawing Cryopreserved Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2.2 First Passage of Cryopreserved Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4.0 Subculture of Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

5.0 Differentiation of Rat Neurospheres into Neurons, Astrocytes and Oligodendrocytes . . . . 6

5.1 Preparation of NeuroCult® NS-A Differentiation Medium (Rat) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65.2 Differentiation of EGF- and/or FGF-b-Responsive Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5.2.1 Preparation of Poly-L-Ornithine Coated Glass Coverslips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65.3 Differentiation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

6.0 Immunolabeling Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

6.1 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86.2 Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86.3 Blocking and Labeling with Primary Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86.4 Secondary Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96.5 Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

7.0 Representative Photographs of Cultured Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . 11

8.0 Representative Photographs of Immunolabeled Differentiated Rat Neurospheres . . . . . . . 13

For Research Use Only

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StemCell TechnologiesVersion 1.1.0

May 2007Catalog #28725




1.0 Materials

1.1 Storage of Medium and Supplements

Very important! Upon arrival, cryopreserved neurospheres* must be stored IMMEDIATELY at -135°C or colder, or in liquid nitrogen.

NeuroCult® NS-A Basal Medium (Rat)* should be stored at 2 - 8°C. NeuroCult® NS-A Proliferation Supplements (Rat)* and NeuroCult®

NS-A Differentiation Supplements (Rat)* should be stored at -20°C.

After NeuroCult® NS-A Proliferation Supplements (Rat) have been added to the NeuroCult® NS-A Basal Medium (Rat), storage at2 - 8°C is recommended for no more than 1 month. Long-term storage at 2 - 8°C is not recommended. Refer to Section 2.0 forpreparation 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 - 8°C is recommended for no more than 1 month. Long-term storage of the"Complete” NeuroCult® NS-A Differentiation Medium (Rat) at 2 - 8°C is not recommended.

1.2 Equipment Required• Vertical laminar flow hood (e.g. Canadian Cabinets) certified for Level II handling of biological materials• Low speed centrifuge (e.g. Beckman TJ-6)• 37°C incubator with humidity and gas control to maintain >95% humidity and an atmosphere of 5% CO2 in air(e.g. Forma 3326)

• Vortex (e.g. Vortex Genie)• Pipette-aid (e.g. Drummond Scientific)• Hemacytometer (e.g. Brightline)• Forceps• Routine light microscope for hemacytometer cell counts• Inverted microscope with flatfield objectives and eye pieces to give object magnification of approximately 20 - 30X, 80X,and 125X (e.g. Nikon Diaphot TMD)

• T-25 cm2 tissue culture flasks (Nunc Catalog #156367 or VWR Catalog #15708-130)• 8-well culture slide (BD BioCoat® 8-well culture slide) pre-coated with Poly-D-Lysine/Laminin (BD Catalog #354688) orPoly-D-Lysine (BD Catalog #354632) OR coverslips (Fisher Scientific Catalog #12-545-82)

• 24-well culture dish (Corning Catalog #3526 or equivalent)• Conical tubes, 14 mL (Falcon Catalog #352001)

1.3 Additional Reagents Required• Recombinant Human Epidermal Growth Factor (rh EGF; Catalog #02633)• Recombinant Human Fibroblast Growth Factor, basic (rh FGF-b; Catalog #02634)• Heparin 0.2% in PBS (Catalog #07980)• Dulbecco's PBS without Ca++ or Mg++ (Catalog #37350)• Trypan Blue (Catalog #07050)• 10 mM Acetic Acid (required for dissolving rh EGF powder)• Bovine Serum Albumin (required for dissolving rh EGF and rh FGF-b powder)• 2% Glucose in PBS• Triton X-100 (Sigma Catalog #T9284)• Poly-L-Ornithine (Sigma Catalog #P3655)• 70% Ethanol• 4% Paraformaldehyde

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* Sold under license from StemCells California, Inc. US Patent Nos. 5,750,376; 5,851,832; 5,980,885; 5,968,829; 5,981,165; 6,071,889; 6,093,531; 6,103,530; 6,165,783; 6,238,922


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2.0 Preparation of “Complete” NeuroCult® NS-A Proliferation Medium (Rat)

2.1 Thawing of Supplements

Bottles of NeuroCult® NS-A Proliferation Supplements (Rat) should be thawed overnight at 2 - 8°C (in the refrigerator) or for 1 - 2hours at 37°C before addition to the NeuroCult® NS-A Basal Medium (Rat). The NeuroCult® NS-A Proliferation Supplements (Rat) canbe aliquoted into 10 mL volumes and stored at -20°C until required for use. However repeated thawing and freezing is notrecommended.

Thaw one bottle (50 mL) of NeuroCult® NS-A Proliferation Supplements (Rat) as described. Add the entire volume of NeuroCult® NS-AProliferation Supplements (Rat) to one bottle (450 mL) of NeuroCult® NS-A Basal Medium (Rat) or add 1 mL of NeuroCult® NS-AProliferation Supplements (Rat) to every 9 mL of NeuroCult® NS-A Basal Medium (Rat) (1/10 dilution).

OR

Add 1 mL of NeuroCult® NS-A Proliferation Supplements (Rat) to every 9 mL of NeuroCult® NS-A Basal Medium (Rat) (1/10 dilution).

2.2 Preparation of Medium, Supplements, and Growth Factors

A stock solution of 10 µg/mL of rh EGF (Catalog #02633; powder at 200 µg/vial) is made up by adding 0.1 mL of sterile 10 mM aceticacid containing at least 0.1% bovine serum albumin (BSA) to initially dissolve the rh EGF powder and then adding 19.9 mL ofNeuroCult® NS-A Basal Medium (Rat) containing NeuroCult® NS-A Proliferation Supplements (Rat). The stock solution of 10 µg/mL of rhEGF should be stored as 0.5 mL aliquots at -20°C until required for use. Add 2 µL of 10 µg/mL rh EGF to every 1 mL of NeuroCult®

NS-A Basal Medium (Rat) containing NeuroCult® NS-A Proliferation Supplements (Rat), to give a final concentration of 20 ng/mL of rhEGF. Mix well.

A stock solution of 10 µg/mL of rh FGF-b (Catalog #02634) is made up in PBS and 0.1% BSA and stored as 0.5 mL aliquots at -20°C.Add 1 µL of rh FGF-b to every 1 mL of NeuroCult® NS-A Basal Medium (Rat) containing NeuroCult® NS-A Proliferation Supplements(Rat) and rh EGF to give a final concentration of 10 ng/mL of rh FGF-b. Mix well.

Store the stock solution of Heparin (Catalog #07980; 0.2% in PBS) at 2 - 8°C in small aliquots (0.5 mL). Add 1 µL of 0.2% Heparinsolution to every 1 mL of NeuroCult® NS-A Basal Medium (Rat) containing NeuroCult® NS-A Proliferation Supplements (Rat), rh EGFand rh FGF-b to achieve a final concentration of 0.0002% (2 µg/mL).

"Complete" NeuroCult® NS-A Proliferation Medium (Rat), containing NeuroCult® NS-A Basal Medium (Rat), NeuroCult® NS-AProliferation Supplements (Rat), 20 ng/mL rh EGF, 10 ng/mL rh FGF-b and 2 µg/mL heparin is now ready to use.

2.2.1 Preparation Summary

For every 1 mL of NeuroCult® NS-A Basal Medium (Rat) containing NeuroCult® NS-A Proliferation Supplements (Rat) add:

Component Volume added per mL Final Concentration10 µg/mL rh EGF 2 µL 20 ng/mL

10 µg/mL rh FGF-b 1 µL 10 ng/mL

0.2% Heparin 1 µL 0.0002% (2 µg/mL)

3.0 Preparation of E18 Rat CNS Cells for In Vitro Culture

3.1 Preparation of Primary Rat CNS Tissue and Initial Culture of Rat Neurospheres

1. Rat embryos (e.g. Sprague/Dawley or Fischer 344) are dissected at gestational day E18, where day E0 is the day agestational plug forms.

2. The brains are removed from the embryos and transferred to a 35 mm plate containing PBS plus 2% glucose, where routinedissection procedures are then performed [Refer to Cell Biology: A Laboratory Handbook. ed. Julio E. Celis. 1998. Volume 1,p149].

3. Dissect out cortex or other desired brain regions and place in PBS containing 2% glucose, on ice.

4. When dissections are complete, transfer tissue in PBS with 2% glucose into a 14 mL conical tube, allow tissues to settle andpipette off supernatant.

5. Resuspend tissue in 1 mL "Complete" NeuroCult® NS-A Proliferation Medium (Rat). Use a 1 mL pipettor with sterile plastic tipto triturate the tissue approximately 5 - 10 times or until single cell suspension is achieved. To triturate, slightly tilt the tip andpress it against the bottom or side of the tube to generate resistance in order to break up the tissue.

Do not introduce air bubbles into the cell suspension. To maintain high viability, avoid using fire-polished glass pipettes todisaggregate neurospheres derived from rat cells.

6. Add 1 mL "Complete" NeuroCult® NS-A Proliferation Medium (Rat) to the single cell suspension and mix carefully to avoidcreating any bubbles. Leave for about 1 minute to allow the undispersed pieces of tissue to settle.

7. Transfer supernatant to a new sterile 14 mL tube. Discard undissociated tissue. Centrifuge supernatant at 150 x g (~800 rpm)for 5 minutes. Discard supernatant.

8. Resuspend cells with a brief trituration (2 times) with a disposable plastic pipette tip in 1 mL "Complete" NeuroCult® NS-AProliferation Medium (Rat).

9. Measure the exact volume and perform a viable cell count on a hemacytometer using a dilution (1/5 or 1/10, depending onamount of tissue dissected) in Trypan Blue (Catalog #07050).

10.Seed cells at a density of 6 x 104 viable cells/mL or 1.2 x 105 viable cells/mL (see note in Section 4.0) in a T-25 cm2 tissueculture flask (Nunc Catalog #156367 or VWR Catalog #15708-130). Use approximately 10 mL of "Complete" NeuroCult® NS-AProliferation Medium (Rat). Incubate cultures in a 5% CO2 incubator at 37°C. Refer to Figures 1 and 2 (Section 7.0) for photosof rat neurospheres o1 and 2 days after plating.

Note: A partial medium change (25 - 30% of total volume) is highly recommended 2 or 3 days after plating, toprevent the medium from becoming acidic. To change the medium, position the flask in an upright position and letthe cells and spheres settle to the bottom (2 - 3 minutes). Slowly remove ~3 mL of medium being careful not toremove the cells, and replace the volume with fresh "Complete" NeuroCult® NS-A Proliferation Medium (Rat).

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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 37°C.

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 andthaw quickly in a 37°C 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 andcentrifuge 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 resuspendneurospheres 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 ofthe neurospheres may not look like intact spheres at this point due to the freeze/thaw process (the morphology shouldrecover the following day). This day (the day the neurospheres are thawed) is considered day 1. Culture in a humidifiedincubator at 37°C and 5% CO2.

8. Check cultures the next day and observe the morphology of the neurospheres. The neurospheres should mostly be floatingsingle 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 inSection 3.2.2.

3.2.2 First Passage of Cryopreserved Rat Neurospheres

1. Pre-wet a 10 mL disposable pipette with “Complete” NeuroCult® NS-A Proliferation Medium (Rat). Remove neurospheres andmedium and place in an appropriately-sized sterile tissue culture tube (e.g. 14 mL tube). Spin at 150 x g (~800 rpm) for 5minutes. Remove supernatant, leaving behind approximately 150 - 180 µL medium. Set the volume of a P200 pipettor withsterile plastic tip and set the volume to slightly less than the approximate volume of the remaining medium (e.g. if the volumeof remaining medium is 180 µL, set the volume of the pipettor to 160 µL to avoid creating bubbles by expelling the entire cellsuspension volume). Pre-wet the tip with medium to reduce cells sticking inside the tip.

2. Gently triturate the cell pellet 10 - 15 times. Slightly tilt the tip and press it against the bottom or side of the tube to generateresistance in order to break up the neurospheres. Rinse the side of the tube during trituration to remove the remainingneurospheres that are attached to the side of the tube. If some neurospheres remain undissociated after 15 triturations (thisusually occurs at later passages), trituration can be extended to a maximum of 25 - 35 times.

To maintain high cell viability avoid using fire-polished glass pipettes to disaggregate neurospheres derived from rat cells.

3. Measure the volume of cells and medium. Count viable cells using Trypan Blue exclusion assay (1/10 dilution) on ahemacytometer.

To avoid passaging cells as clumps, pipette cells up and down with a P200 pipette tip 4 - 8 times to obtain single cellsuspension prior to seeding cells in a flask.

4. Seed cells at densities of 1.25 x 104 and 2.5 x 104 viable cells/mL in a T-25 cm2 tissue culture flask (Nunc Catalog #156367 orVWR Catalog #15708-130) containing 10 mL of "Complete" NeuroCult® NS-A Proliferation Medium (Rat).

Two different seeding densities should be used to ensure that an appropriate number of cells are cultured for efficientneurosphere formation.

5. Incubate cultures in a 5% CO2 incubator at 37°C.

6. Cultures should be examined under the microscope regularly. A partial medium change should be performed 2 days after thecultures are set up.

4.0 Subculture of Rat Neurospheres

1. Observe the neurosphere cultures under a microscope to determine if the neurospheres are ready for passaging. Cultures ofrat E18 cortical or striatal neurospheres should be passaged every 3 - 4 days. Neurospheres should be passaged before thediameter exceeds 100 µm to avoid hypoxic cells in the centre of the spheres. Refer to Figure 3 (Section 7.0), for a photo ofneurospheres ready to be passaged.

Cells will proliferate as spheroids, called neurospheres, which in general detach from the surface of the tissue culture flaskand float in suspension. The neurospheres should be ready for subculture 3 - 4 days after plating depending on spheredensity and size. Viable neurospheres will, for the most part, be semi-transparent phase contrast bright, with many of thecells on the outer surface displaying microspikes.

2. Remove neurospheres and medium and place in an appropriately-sized sterile tissue culture tube (e.g. 14 mL tube). Spin at90 x g (~400 rpm), for 5 minutes. Remove supernatant, leaving behind approximately 150 - 180 µL medium. Set the volumeof a P200 pipettor with sterile plastic tip to slightly less than the approximate volume of the remaining medium (e.g. if thevolume of remaining medium is 180 µL, set the volume of the pipettor to 160 µL to avoid creating bubbles by expelling theentire cell suspension volume). Pre-wet the tip with medium to reduce cells sticking inside the tip.

3. Gently triturate the cell pellet 10 - 15 times. Slightly tilt the tip and press it against the bottom or side of the tube to generateresistance in order to break up the neurospheres. Rinse the side of the tube during trituration to remove the remainingneurospheres that are attached to the side of the tube. If some neurospheres remain undissociated after 15 triturations (thisusually occurs at later passages) trituration can be extended to a maximum of 25 - 35 times.

To maintain high cell viability, avoid using fire-polished glass pipette to disaggregate the neurospheres.

4. Measure the volume of cells and medium. Count viable cells using Trypan Blue exclusion assay (1/10 dilution at earlierpassages, lower dilution for later passages) on a hemacytometer.

To avoid passaging cells as clumps, pipette cells up and down with a P200 pipette tip 4 - 8 times to obtain single cellsuspension prior to seeding cells in a flask.

5. Seed cells at a density of 1.25 x 104 or 2.5 x 104 viable cells/mL (see note below) in a T-25 cm2 tissue culture flask (NuncCatalog #156367 or VWR Catalog #15708-130) containing 10 mL of "Complete" NeuroCult® NS-A Proliferation Medium (Rat).

6. Incubate cultures in a 5% CO2 incubator at 37°C.

7. Cultures should be examined under the microscope regularly. A partial medium change should be performed at Day 2 afterculture set-up as described in Section 3.1, Step 10.

Important Note:

Neural stem and progenitor cells from rat CNS tissue have different growth characteristics compared to mouse neural stemand progenitor cells. The rate of proliferation of E18 rat cortical is higher (varies from 2 - >20 fold expansion of total cellsevery 3 - 4 days of culture) during early passages (P2 to P5). A critical phase in the growth curve occurs between P5 and P9(20 - 36 days) in culture, when a significant decrease in terms of fold expansion (average between 2 - 5 fold) is observed.During this critical period, cells start to attach to the flask and differentiate. To reduce attachment of spheres to the flask, it isimportant to perform partial medium changes every two days and be careful not to disrupt the attached cells so that only thefloating spheres are passaged. It is suggested that more than one flask is cultured during the critical passages, P5 - P9. Twodifferent seeding densities, 1.25 x 104 cells/mL and 2.5 x104 cells/mL, should be used to ensure a sufficient number of cellsare obtained for the next passage.

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5.0 Differentiation of Rat Neurospheres into Neurons, Astrocytes andOligodendrocytes

In the presence of rh EGF and rh FGF-b, rat neural stem cells and their progeny within neurospheres remain in a relativelyundifferentiated state. Upon removal of the growth factors and addition of a small amount of serum, differentiation to neurons,astrocytes and oligodendrocytes is induced.

5.1 Preparation of NeuroCult® NS-A Differentiation Medium (Rat)

Thaw one bottle containing 50 mL of NeuroCult® NS-A Differentiation Supplements (Rat) at room temperature or overnight at 2 - 8°C.The NeuroCult® NS-A Differentiation Supplements (Rat) can be aliquoted into 10 mL volumes and stored at -20°C. However repeatedfreezing and thawing is not recommended.

Add the entire volume (50 mL) of NeuroCult® NS-A Differentiation Supplements (Rat) to the bottle (450 mL) of NeuroCult® NS-A BasalMedium (Rat) or add 1 mL of NeuroCult® Differentiation Supplements (Rat) to every 9 mL of NeuroCult® NS-A Basal Medium (Rat)(1/10 dilution).

The "Complete" NeuroCult® NS-A Differentiation Medium (Rat) is now ready for use.

5.2 Differentiation of EGF- and/or FGF-b-Responsive Rat Neurospheres

To initiate differentiation, single cells (dissociated from neurospheres) are cultured on commercially available 8-well culture slides (BDBioCoat® pre-coated with Poly-D-Lysine/Laminin Catalog #354688, or Poly-D-Lysine Catalog #354632) or on Poly-L-Ornithine coatedround glass coverslips (refer to Section 5.2.1).

5.2.1 Preparation of Poly-L-Ornithine Coated Glass Coverslips

1. If using round glass coverslips (Fisher Scientific #12-545-82) for immunolabeling, soak coverslips in 70% ethanol andindividually hand clean with a lint-free tissue. Sterilize ethanol-cleaned coverslips by autoclaving prior to coating. Coatcoverslips for 24 hours prior to use.

2. Using sterile forceps, transfer a single sterile glass coverslip to a well of a 24-well plate.

3. Dispense 1 mL of 15 µg/mL Poly-L-Ornithine solution (Sigma Catalog #P3655) into each well.

4. Incubate glass coverslips for a minimum of 3 hours at 37°C. After the incubation, remove the Poly-L-Ornithine solution fromeach well by aspiration and rinse each well 3 x 15 minutes with sterile PBS (Catalog #37350).

5. The pre-coated Poly-L-Ornithine glass coverslips are now ready for the differentiation assay (Section 5.3).

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-LysineCatalog #354632) add 0.75 mL/well of "Complete" NeuroCult® NS-A Differentiation Medium (Rat). If using Poly-L-Ornithinecoated glass coverslips prepared as described in Section 5.2.1, place a single prepared coated coverslip into an individualwell of a 24-well culture dish (e.g. Corning Catalog #3526) containing 1 mL/well of "Complete" NeuroCult® NS-ADifferentiation Medium (Rat).

2. After 3 - 4 days of culturing rat neurospheres in "Complete" NeuroCult® NS-A Proliferation Medium (Rat), remove the mediumwith 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 µL "Complete" NeuroCult® NS-A Differentiation Medium (Rat). With a P200 pipettetip, triturate the neurosphere suspension until single cell suspension is achieved (refer to Section 4, Steps 2 and 3 for adescription of trituration).

5. Resuspend the single cell suspension with 10 mL of “Complete” NeuroCult® NS-A Differentiation Medium (Rat). Spin at150 x g (~800 rpm) for 5 minutes. Remove all of the supernatant and discard.

6. Resuspend the cell pellet in approximately 200 µL 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 platedwith 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-wellchamber 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. Ifthe medium becomes acidic (yellow), a half-medium change should be performed by removing approximately 50% of themedium 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 andprocessed immediately for indirect immunofluorescence as described in Section 6.0.

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6.0 Immunolabeling Procedure

6.1 Fixation

1a.If using cells grown on pre-coated 8-well chamber slides, remove the culture medium from each chamber containingdifferentiated cells (taking care not to remove all the medium to avoid exposure of the unfixed cells to air) and add 0.5 mL ofthe 4% paraformaldehyde solution directly into the chamber. Incubate for 30 minutes at room temperature.

OR

1b.If using cells grown on coverslips, add 1 mL of 4% paraformaldehyde (in PBS pH 7.2) to a new 24-well plate. Transfercoverslips containing differentiated cells into the paraformadehyde solution (one coverslip/well, cells facing up). Fix cells in4% paraformaldehyde by incubation at room temperature for 30 minutes.

2. Aspirate the paraformaldehyde solution. For ease, an aspiration system connecting to a vacuum pump may be used.

3. Add PBS (pH 7.2) to the samples and incubate for 5 minutes. Repeat this washing procedure two more times for a total of 3wash steps (aspiration with a vacuum pump is used each time to remove the supernatant).

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 for5 - 10 minutes at room temperature.

2. Remove PBS/Triton-X 100 by aspiration. Perform 2 x 5 minute PBS washes.

6.3 Blocking and Labeling with Primary Antibodies

1. Make up a solution of PBS with 10% serum. This will be used as the diluent for the primary antibody. The type of serum useddepends on the secondary antibody chosen. Refer to Table 1 (below) to choose serum that is appropriate for differentsecondary antibodies.

Table 1: Appropriate primary antibody blocking serum for different conjugated secondary antibodies

Secondary Antibody Serum Catalog #Affini-Pure Goat anti-mouse IgM, µ chain specific FITC-conjugated goat 10211Affini-Pure Goat anti-rabbit IgG, (H+L) FITC-conjugated goat 10212

Affini-Pure Goat anti-mouse IgG, (H+L) Texas Red dye-conjugated goat 10213

Affini-Pure Goat anti-rabbit IgG, (H+L) AMCA-conjugated goat 10214

2. Dilute primary antibody in the appropriate serum-containing diluent (from Table 1). Appropriate working dilutions forimmunolabeling are shown in Table 2 (below).

Table 2: The optimal working dilution used for different primary antibodies

The working dilutions are only recommendations as the optimal working dilution for each specific application should bedetermined by the user.

3. Add diluted primary antibodies to the 24-well plate or chamber slides in a minimum volume of 250 µL. Alternately place asmall volume of antibody, approximately 50 µL, directly on the coverslip containing the differentiated cells and place a cleansecond coverslip directly on top. Place in a hydrating chamber.

4. Incubate for 2 hours at 37°C or overnight at 2 - 8°C.

5. Wash off primary antibody with 3 x 5 minute washes using PBS.

6.4 Secondary Staining

1. Dilute secondary antibodies 1/100 in PBS + 2% serum (same serum used as diluent for the primary antibody, Table 1). Add aminimum volume of 250 µL to 24-well plate or chamber slide.

2. Incubate secondary antibodies for 30 minutes at 37°C.

Note: Secondary antibody is sensitive to light and therefore keep samples in the dark whenever possible to prevent bleaching.

3. Wash off secondary antibody with 3 x 5 minute washes using PBS.

4. After the last wash add distilled water to each well.

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Targeted Antigen Clone Isotype Catalog # Working DilutionGABA - rabbit polyclonal 01411 1:200

Glial Fibrillary Acidic Protein (GFAP) - rabbit polyclonal 01415 1:100

Microtubule Associated Protein-2 (MAP2) AP20 mouse IgG1 01410 1:200

Myelin Basic Protein (MBP) - rabbit polyclonal 01417 1:200

Nestin Rat 401 mouse IgG1 01418 1:50

Neuronal Class III β-Tubulin TUJ1 mouse IgG2a 01409 1:1000

Oligodendrocyte Marker O4 O4 mouse IgM 01416 1:50

Oligodendrocyte Marker RIP RIP mouse IgG1 01433 1:100

Tyrosine Hydroxylase-2 TH2 mouse IgG1 01412 1:400



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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 glassslides. Rinse slides in distilled water in a coplin jar.

2. Add about 5 µL of mounting medium (e.g. FluorSave™ Reagent, Calbiochem Catalog #345789) in each chamber slot and coverwith a 75 mm coverslip avoiding trapping any air bubbles.

3. If coverslips are used, add 10 µL of mounting medium (e.g. FluorSave™ Reagent, Calbiochem Catalog #345789) to a cleanglass coverslip. Remove immunostained coverslip from the 24-well plate and gently tap corner of the coverslip to removeexcess 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). Referto 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 Absorption Peak (nm) Emission Peak (nm)Aminomethylcoumarin, AMCA 350 450Fluorescein Isothiocyanate, FITC 492 520

Rhodamine Red-X, RRX 570 590

Texas Red, TR 596 620

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Catalog #28725




7.0 Representative Photographs of Cultured Rat Neurospheres

Figure 1. Small neurospheres, one day after platingsingle cell suspensions of rat E18 cortex in “Complete”NeuroCult® NS-A Proliferation Media (Rat).

Magnification: 125X

Figure 2. Neurospheres derived from rat E18 corticalcells after 2 days of culture in “Complete” NeuroCult®

NS-A Proliferation Medium (Rat). The size (diameter) ofthe neurospheres has increased.

Magnification: 125X

Figure 3. Neurospheres derived from rat E18 corticalcells after 4 days in culture with “Complete” NeuroCult®

NS-A Proliferation Medium (Rat). Neurospheres areready to be passaged. If not passaged, cells in thecenter of the spheres will become dark and hypoxic andthe spheres will stick down to the bottom of flask.

Magnification: 125X


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Figure 4. The critical phase in rat neuropsherescultures: passages 5 - 9 (20 - 36 days in culture). At thispoint some neurospheres will adhere to the flask andstart differentiating. It is important to perform a partialmedium change every other day and passage only thefloating neurospheres (to prevent differentiation).

Magnification: 125X

Figure 5. Neurospheres derived from rat E18 corticalcells which have all adhered and differentiated after 3days in culture. The medium should be regularlyreplenished and only floating neurospheres passaged toprevent cells attaching to the flask.

Magnification: 125X

Figure 6. “Unhealthy” neurospheres derived from ratE18 cortical cells after 4 days in culture. Individualneurospheres are beginning to dissociate into singlecells in culture, an indication of a problem with theculture conditions or cells.

Magnification: 125X

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Catalog #28725




Figure 7. Differentiated rat neural cells cultured with NeuroCult® NS-A Differentiation Medium (Rat). Cells are immunostained with lineagespecific antibodies as described in Section 6.3 and counterstained with DAPI (blue).

A. β-Tubulin (red) and MBP (Green)B. Oligodendrocyte Marker O4 (green) and β-Tubulin (red)C. Nestin (red)D. GFAP (green) and MAP2 (red)E and F. Triple staining with Oligodendrocyte Marker O4 (green), GFAP (blue) and β-Tubulin (red)

Magnification: 400X

A B

C D

E F

8.0 Representative Photographs of Immunolabeled Differentiated Rat Neurospheres



Catalog #28725




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