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JINGLY FUNG et al.
B. Metaphase spread slides are pretreated following the instructions of
the SKY kit
1. Look at the slide under phase contrast and select a region of interest
containing at least five metaphase spreads and mark area with a diamond tip pen.
2. Incubate slides in the pepsin solution (I5 IJI stock pepsin solution in
50 ml of 0.01 M HCI solution) at 37°C for 3-5 min.
3. Wash slides twice in 1x PBS at room temperature for 5 min each.
4. Wash slides in 1xPBS/MgCh at room temperature for 5 min.
5. Incubate slides in solution of 1% formaldehyde in PBS/MgCh at room
temperature for 10 min.
6. Wash slides in 1xPBS at room temperature for 5 min.
7. Dehydrate slides in 70%,80%, 100% ethanol for 3 min each at room
temperature.
8. Air dry slides at room temperature.
9. Look at the slide under phase-contrast, and note any remaining cytoplasm, debris, etc.
Interphase cells
Human blastomeres All procedures followed protocols approved by the UCSF Committee on
Human Research for using embryos for research. Prior written consent
was obtained from all donors.
Caution: throughout the entire procedure, the embryos and blastomeres are observed under a microscope.
1. Incubate the donated embryos in 0.5% pronase/FHM to remove the
zona pellucida.
2. Transfer the embryos into Ca2+/Mg
2
+-free PBS.
3. Separate the blastomeres by repeated pipetting of the embryo.
4. Transfer blastomeres to 1x PBS.
5. Incubate individual blastomeres in a hypotonic solution of 1% Nacitrate, 6 mg/ml bovine serum albumin in water for 5 min.
JINGLY FUNG et al.
B. Metaphase spread slides are pretreated following the instructions of
the SKY kit
1. Look at the slide under phase contrast and select a region of interest
containing at least five metaphase spreads and mark area with a diamond tip pen.
2. Incubate slides in the pepsin solution (I5 IJI stock pepsin solution in
50 ml of 0.01 M HCI solution) at 37°C for 3-5 min.
3. Wash slides twice in 1x PBS at room temperature for 5 min each.
4. Wash slides in 1xPBS/MgCh at room temperature for 5 min.
5. Incubate slides in solution of 1% formaldehyde in PBS/MgCh at room
temperature for 10 min.
6. Wash slides in 1xPBS at room temperature for 5 min.
7. Dehydrate slides in 70%,80%, 100% ethanol for 3 min each at room
temperature.
8. Air dry slides at room temperature.
9. Look at the slide under phase-contrast, and note any remaining cytoplasm, debris, etc.
Interphase cells
Human blastomeres All procedures followed protocols approved by the UCSF Committee on
Human Research for using embryos for research. Prior written consent
was obtained from all donors.
Caution: throughout the entire procedure, the embryos and blastomeres are observed under a microscope.
1. Incubate the donated embryos in 0.5% pronase/FHM to remove the
zona pellucida.
2. Transfer the embryos into Ca2+/Mg
2
+-free PBS.
3. Separate the blastomeres by repeated pipetting of the embryo.
4. Transfer blastomeres to 1x PBS.
5. Incubate individual blastomeres in a hypotonic solution of 1% Nacitrate, 6 mg/ml bovine serum albumin in water for 5 min.