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Fig. 6 Interaction of a motor neuron growth cone with an extrinsic cue. An individual motor neuron growth cone encounters the extrinsic cue tumor necrosis factor a, coupled covalently onto a small polystyrene bead (5 ^m in diameter). Upon intial filopodia contact, the growth cone displays changes in behavior and morphology over a time period of 30 min. Finally the growth cone collapses, which is indicated by a complete loss of morphology and cessation of advance.

Fig. 6 Interaction of a motor neuron growth cone with an extrinsic cue. An individual motor neuron growth cone encounters the extrinsic cue tumor necrosis factor a, coupled covalently onto a small polystyrene bead (5 ^m in diameter). Upon intial filopodia contact, the growth cone displays changes in behavior and morphology over a time period of 30 min. Finally the growth cone collapses, which is indicated by a complete loss of morphology and cessation of advance.

neurons is accomplished using defined medium with Leibovitz' L15 medium replacing DMEM. Leibovitz' L15 medium (Gibco) is phosphate buffered and thus maintains a pH of 7.4 irrespective of room atmosphere. A heating system is required to maintain cultures on the microscope table at 37° C.

IX. Preparation of Solutions, Culture Dishes, Media, and Media Supplements

1. Sterilization Procedures

Sterilize all glassware by autoclaving (20min, 121°C, 1 bar). Label all items with autoclave indicator tape. Sterilize large volumes of solutions (100 ml or more) by filtration over 0.22-ym filter membranes using disposable bottle-top filters. Small volumes of solutions are best sterilized using disposable syringes and 0.22-ym syringe filters.

2. Glass Coverslips

Insert glass coverslips in a holding device (22 x 22 mm2, #1, German glass, Carolina Biological Supply Company, Burlington, NC). Teflon holders or wafer baskets used to wash computer chips work well (Entegris Inc., Chaska, MN, Wash coverslips in 2% Micro-90 (VWR, 10 ml detergent per

500 ml water) heated to 50-55° C for 30min. Rinse glass coverslips 10 times with double distilled water and twice with MilliQ water. Place glass coverslips onto a filter paper (Whatman #1, Millipore) to dry in a convection oven at 65° C overnight. Sterilize washed coverslips under ultraviolet light (UV) for 15 min in a laminar flow hood and store at room temperature indefinitely in a sterile container. Coverslips can be either glued onto the bottom of drilled dishes or inserted into a 35-mm tissue culture dish.

3. Drilled Dishes

Drill a 1-cm-diameter hole into the bottom of 35-mm tissue culture dishes using a drill press equipped with an 11/16-in. drill bit. For protection, wear gloves and goggles. Clean debris from hole and smooth rims using a scalpel. Apply aquarium glue (local pet store) around the hole using a 1-ml disposable syringe. Firmly press a washed glass coverslip over the hole and dry dishes at room temperature over night (dishes can be stored indefinitely). For sterilization, expose dishes to UV light for 15 min in a laminar flow hood.

4. Hanks' Balanced Salt Solution

HBSS is a phosphate buffer that maintains pH 7.4 in normal air. HBSS (Gibco) is purchased as a 10 x sterile liquid and is stored at room temperature. Dilute 1 volume of 10 x HBSS with 9 volumes of sterile MilliQ water. Store 1x HBSS (working solution) at 4° C.

5. Cell Dissociation Solution

Cell dissociation solution (Gibco, 10 x stock solution) contains 0.5% trypsin and 5.3 mM Na4EDTA. Prepare and store aliquots (1ml and 100 ^l) at —20° C.

Dissolve DNase I (200 mg, Roche Diagnostics Corp., Indianapolis, IN) in HBSS (10 ml) and store aliquots (0.5 ml) -20° C.

7. Horse Serum and Fetal Bovine Serum

Thaw horse serum (Hyclone, Logan, UT), or fetal bovine serum (Hyclone) at 4° C. Gently swirl and heat inactivate liquid serum by placing in a 56° C warm water bath for 30 min. After cooling in a laminar flow hood, prepare 5- and 10-ml aliquots and store aliquots at —20° C.

8. DMEM (High Glucose, Gibco)

Dissolve contents of one bag in 900 ml MilliQ water, add 3.7 g NaHCO3, and adjust pH to 7.4. Adjust volume to 1 liter with MilliQ water, sterilize by filtration over a 0.22-^m membrane, and store at 4° C.

Mix 10 ml heat-inactivated FBS with 90 ml DMEM, sterilize by filtration through 0.22-ym membranes, and store at 4° C.

10. 100 X N3 Supplement

Prepare aliquots of each component (100x stock solutions) and store at —20° C without prior sterilization by filtration over 0.22-ym membranes.

10mg/ml BSA fraction V (Sigma) in HBSS (500-yl aliquots)

100mg/ml Apo-transferrin human (Calbiochem) in HBSS (500-yl aliquots)

25 mg/ml insulin (Sigma) in 0.01 M HCI (200-yl aliquots)

80.55 yg/ml putrescin (Sigma) in HBSS (200-yl aliquots)

200 yg/ml tri-iodothyronin (Sigma) in 0.01 M NaOH (50-yl aliquots)

10.4 yg/ml selenium (Sigma) in HBSS (500-yl aliquots)

125.8 yg/ml progesterone (Sigma) in absolute ethanol (50-yl aliquots)

2 mg/ml corticosterone (Sigma) in absolute ethanol (50-yl aliquots)

For a 100x stock of the N3 supplement, place 2ml HBSS in a 15-ml conical tube and add 1 aliquot of each component. Mix gently by swirling the tube and adjust the volume to 5 ml with HBSS. Prepare N3 aliquots (500 yl and 1 ml) and store at 20° C.

11. 100 X 5-Fluoro-2'-deoxyuridme (FdUr)

Dissolve 29.544 mg FdUr (Sigma) and 73.26 mg uridine (Sigma) in 10ml HBSS. Prepare 500-yl and 1-ml aliquots and store at —20° C.

12. Spinal Cord Medium

Add 5ml FBS and 0.5ml of each 100x N3 supplement and 100x FdUr to DMEM for a total volume of 50 ml. Sterilize by filtration over a 0.22-ym membrane using a syringe filter and a 50-ml disposable syringe. Store at 4° C.

13. Defined Medium

Dissolve 50 mg BSA (fraction V) in 49 ml DMEM. Add 0.5 ml of each 100 x N3 supplement and 100 x FdUr and sterilize by filtration over a 0.22-ym membrane using a syringe filter and a 50-ml disposable syringe. Store at 4° C. For live video observations, replace DMEM with Leibovitz' L15 (Gibco).

14. Substrate Coating

Dissolve 1.24 g boric acid (Sigma) in 400 ml MilliQ water. Dissolve 1.9 g tetrasodium borate (Sigma), adjust pH to 8.4, and store buffer at 4°C. Add 1mg poly-D-lysine (Sigma) to 10 ml borate buffer (100 yg/ml) and store 0.5-ml aliquots at —20° C. Apply 500 yl poly-D-lysine solution onto glass coverslip for each drilled dish and incubate for 30 min at room temperature. Rinse dishes three times with MilliQ water and air dry in a laminar flow hood. Sterilize dishes with UV light for 15 min. Dishes can be stored at room temperature indefinitely. Laminin (Roche Diagnostics Corp., a 1-mg sterile solution) is prepared in aliquots (1 yg/yl, 100 yl) at —20° C. For coating, apply 20 yl laminin for each poly-D-lysin-treated glass coverslip and add 200 yl HBSS. Incubate dishes for at least 2h in a CO2 incubator. Prior to plating cells, rinse glass coverslips with 500 yl HBSS.

Add 875 mg BSA to 25 ml DMEM. Simply allow the BSA to go into solution (no vortexing). Sterilize over a 0.22-ym filter membrane using a syringe filter. Store 5-ml aliquots at —20° C.

16. 6.4% Metrizamide

Dissolve 6.4 g of metrizamide (Sigma) in 10 ml MilliQ water and adjust volume to 20 ml. Store this solution (32% metrizamide) at 4° C. Prior to use, place 1 ml of 32% metrizamide into a 15-ml conical centrifuge tube and add 4 ml DMEM. Mix well and keep on ice until use.

17. Web Sites

A plethora of useful information about developmental biology can be found at the following web sites: Zygote (, Gilbert-Developmental Biology (, Society for Developmental Biology (http://, and Virtual Embryo (


Research related to this manuscript was supported in part by The Christopher Reeve Paralysis Foundation (Grant KAC1-0004), The Special Neuroscience Research Program (1U54NS41069), and NCRR COBRE Grant RR15583.


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