chick trunk neural tube and examining sites in the periphery to
which their progeny migrate, we discovered a more complex, unordered emigration of trunk neural crest cells than previously thought
[2]. Our extensive experience with in vitro slice culture and whole
embryo imaging in chick and quail embryos allowed us to define
the conditions required for single cell resolution of fluorescently
labeled cells during the epithelial-to-mesenchymal transition of the
trunk neural crest cells. Here, we present a method to culture whole
chick embryo explants and maintain embryo orientation and health
over a 24 h period. Although our approach described here is an
exciting step forward in allowing direct observation and measurement of complex cell behaviors during trunk neural crest cell
epithelial-to-mesenchymal transition, our methods may be applicable for a wider range of phenomena and in other research
organisms.
2 Materials
2.1 Embryology
and Labeling
1. Fertilized chicken eggs.
2. DiI labeling solution: 50 μg DiI powder (C7000, Thermo
Fisher, or equivalent), 10 μL ethanol, 90 μL 0.3 M sucrose.
3. Microinjector (Picospritzer, Parker Hannifin, or equivalent).
4. Glass needles pulled to a shape you prefer (BF100-50-10,
Sutter Instrument, or equivalent).
5. Whatman filter paper.
6. Embryology tools (forceps, tungsten needle with holder, small
scissors).
7. Ink Solution: Sterilized 10% black pen ink in PBS.
8. 10 mL and 1 mL syringes.
9. 18- and 25-gauge needles.
10. Ringer’s Solution: 7.2 g Sodium Chloride, 0.37 g Potassium
Chloride, 0.17 g Calcium Chloride per liter, pH 7.4.
11. Phosphate Buffered Saline pH 7.4.
12. Optional: fluorescent protein vector(s) and a square wave electroporator (ECM 830, BTX; or equivalent).
2.2 Mounting
1. Approximately 2.5 cm diameter coin and pencil.
2. 60 mm plastic petri dishes.
3. EC Culture Medium: egg albumen, agar solidifying agent
(Bacto Agar, 214050, BD Diagnostics, or equivalent), Penicillin–Streptomycin, PBS.
4. 49
water bath or similar.
108
Mary Cathleen McKinney and Paul M. Kulesa
which their progeny migrate, we discovered a more complex, unordered emigration of trunk neural crest cells than previously thought
[2]. Our extensive experience with in vitro slice culture and whole
embryo imaging in chick and quail embryos allowed us to define
the conditions required for single cell resolution of fluorescently
labeled cells during the epithelial-to-mesenchymal transition of the
trunk neural crest cells. Here, we present a method to culture whole
chick embryo explants and maintain embryo orientation and health
over a 24 h period. Although our approach described here is an
exciting step forward in allowing direct observation and measurement of complex cell behaviors during trunk neural crest cell
epithelial-to-mesenchymal transition, our methods may be applicable for a wider range of phenomena and in other research
organisms.
2 Materials
2.1 Embryology
and Labeling
1. Fertilized chicken eggs.
2. DiI labeling solution: 50 μg DiI powder (C7000, Thermo
Fisher, or equivalent), 10 μL ethanol, 90 μL 0.3 M sucrose.
3. Microinjector (Picospritzer, Parker Hannifin, or equivalent).
4. Glass needles pulled to a shape you prefer (BF100-50-10,
Sutter Instrument, or equivalent).
5. Whatman filter paper.
6. Embryology tools (forceps, tungsten needle with holder, small
scissors).
7. Ink Solution: Sterilized 10% black pen ink in PBS.
8. 10 mL and 1 mL syringes.
9. 18- and 25-gauge needles.
10. Ringer’s Solution: 7.2 g Sodium Chloride, 0.37 g Potassium
Chloride, 0.17 g Calcium Chloride per liter, pH 7.4.
11. Phosphate Buffered Saline pH 7.4.
12. Optional: fluorescent protein vector(s) and a square wave electroporator (ECM 830, BTX; or equivalent).
2.2 Mounting
1. Approximately 2.5 cm diameter coin and pencil.
2. 60 mm plastic petri dishes.
3. EC Culture Medium: egg albumen, agar solidifying agent
(Bacto Agar, 214050, BD Diagnostics, or equivalent), Penicillin–Streptomycin, PBS.
4. 49
water bath or similar.
108
Mary Cathleen McKinney and Paul M. Kulesa