2.1.2 Microdissection
(Fig. 1)
1. Hair knife. Dip the tip of a short glass Pasteur pipette into
melted wax and insert the root of a hair (eyebrow, eyelash or
else) in the tip of the pipette before the wax has set.
2. A dish filled with modelling clay.
3. Glass ball. Prepared by melting the tip of a short glass Pasteur
pipette.
4. Fine forceps.
5. P20 micropipette.
6. A small uncoated petri dish filled with culture medium to
collect NC explants.
2.1.3 Amphibian Media
and Culture Media
1. Normal Amphibian Medium (NAM) stock solutions, must be
autoclaved and can be stored at room temperature. In distilled
water, NAM A (NaCl, 1.1 M; KCl, 20 mM; NaHCO 3 ,
10 mM), NAM B (MgSO 4 , 10 mM; CaCl 2 , 10 mM), NAM
C (EDTA, 1 mM), NAM D (Na 2 HPO 4 , 16 mM; NaH 2 PO 4 ,
4 mM).
2. Working solutions: NAM 0.1Â (for 1 L add 10 mL of each
NAM stock solutions and add 960 mL of distilled water), NAM
0.25Â (for 1 L add 25 mL of each NAM stock solutions and
add 900 mL of distilled water).
3. Culture medium—Danilchik’s for Amy (DFA): 53 mM NaCl,
5 mM Na 2 CO 3 , 4.5 mM KGluconate, 32 mM NaGluconate,
1 mM MgSO 4 (7H 2 O), 1 mM CaCl 2 , 0.1% BSA; adjust pH to
8.3 with 1 M Bicine. Store at À20
C. After thawing, add
1000 U penicillin and 100 μg/mL streptomycin.
2.2 Time-Lapse
Imaging
1. Microscopes: inverted, upright microscope, dissecting microscope (see Note 1).
2. Camera and software.
3. Temperature controlled room (see Note 2).
4. Formaldehyde.
2.3 Analysis of Cell
Migration
and Dispersion
Dynamics
1. A computer equipped with a modern CPU and with sufficient
amount of RAM and storage space to process the data. The
total RAM available should be twice the size of your largest
dataset to allow opening and processing of the data.
2. A free (FIJI/Image J) or commercial software (e.g., Imaris/
Bitplane) for automated detection of nuclei, tracking, and
dispersion analysis.
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(Fig. 1)
1. Hair knife. Dip the tip of a short glass Pasteur pipette into
melted wax and insert the root of a hair (eyebrow, eyelash or
else) in the tip of the pipette before the wax has set.
2. A dish filled with modelling clay.
3. Glass ball. Prepared by melting the tip of a short glass Pasteur
pipette.
4. Fine forceps.
5. P20 micropipette.
6. A small uncoated petri dish filled with culture medium to
collect NC explants.
2.1.3 Amphibian Media
and Culture Media
1. Normal Amphibian Medium (NAM) stock solutions, must be
autoclaved and can be stored at room temperature. In distilled
water, NAM A (NaCl, 1.1 M; KCl, 20 mM; NaHCO 3 ,
10 mM), NAM B (MgSO 4 , 10 mM; CaCl 2 , 10 mM), NAM
C (EDTA, 1 mM), NAM D (Na 2 HPO 4 , 16 mM; NaH 2 PO 4 ,
4 mM).
2. Working solutions: NAM 0.1Â (for 1 L add 10 mL of each
NAM stock solutions and add 960 mL of distilled water), NAM
0.25Â (for 1 L add 25 mL of each NAM stock solutions and
add 900 mL of distilled water).
3. Culture medium—Danilchik’s for Amy (DFA): 53 mM NaCl,
5 mM Na 2 CO 3 , 4.5 mM KGluconate, 32 mM NaGluconate,
1 mM MgSO 4 (7H 2 O), 1 mM CaCl 2 , 0.1% BSA; adjust pH to
8.3 with 1 M Bicine. Store at À20
C. After thawing, add
1000 U penicillin and 100 μg/mL streptomycin.
2.2 Time-Lapse
Imaging
1. Microscopes: inverted, upright microscope, dissecting microscope (see Note 1).
2. Camera and software.
3. Temperature controlled room (see Note 2).
4. Formaldehyde.
2.3 Analysis of Cell
Migration
and Dispersion
Dynamics
1. A computer equipped with a modern CPU and with sufficient
amount of RAM and storage space to process the data. The
total RAM available should be twice the size of your largest
dataset to allow opening and processing of the data.
2. A free (FIJI/Image J) or commercial software (e.g., Imaris/
Bitplane) for automated detection of nuclei, tracking, and
dispersion analysis.
260
Nade ` ge Gouignard et al.