2 Materials
2.1 General
Materials
1. Inverted microscope equipped with micromanipulation and
aspiration system.
2. Stereomicroscope with thermal plate, for maintaining materials
at 37
C.
3. Holding pipettes with a 25–30/120–150 μm inner/outer
diameter (e.g., MPH-XLG from Origio).
4. Aspiration micropipettes with a 17–10 μm inner diameter and
beveled opening (e.g., MSC-18-30 from Origio).
5. Low rim Petri dishes (as used for micromanipulation).
6. Mineral oil, suitable for mammalian embryo culture.
7. Four-well dishes.
8. 0.5 and 0.25 mL straws and 0.25 mL hemistraw (see Note 1).
Hemistraws can be prepared, under sterile conditions, using a
scalpel and 0.25 mL straw (see Fig. 2c).
9. Straw connector.
10. Labeling system for 0.5 mL straws.
11. Pipette for handling small volumes (2–10 μL), with sterile tips.
12. Stripper pipette, with stripper tips 125 μm in diameter (MXL3125, Origio).
13. Styrofoam box containing liquid nitrogen (see Note 2).
14. Fine tweezers.
15. Handling medium. PBS with no calcium or magnesium, 0.4%
(w/v) BSA and 0.1% (w/v) PVP360.
2.2 Holding Medium,
Vitrification, and
Warming Solutions
1. Holding medium (HM). Prepare by supplementing PBS with
20% fetal bovine serum (FBS) and 5 μg/mL gentamicin. Filter
and keep in the refrigerator for no longer than a week.
2. Vitrification solution 1 (VS1). Prepare in HM: 7.5% (v/v)
ethylene glycol and 7.5% (v/v) dimethyl sulfoxide (DMSO)
(see Note 3). Filter-sterilize, prepare 0.5 mL aliquots, and
freeze.
3. Vitrification solution 2 (VS2). Prepare in HM: 16.5% v/v EG,
16.5% v/v DMSO, and 0.5 M sucrose. Filter-sterilize, prepare
0.5 mL aliquots, and freeze.
4. Warming solution 1 (WS1). Dissolve in HM: 0.33 M sucrose.
Filter-sterilize, prepare 1 mL aliquots, and freeze.
5. Warming solution 2 (WS2). Dissolve in HM: 0.2 M sucrose.
Filter-sterilize, prepare 0.5 mL aliquots, and freeze.
518
Carolina Herrera
2.1 General
Materials
1. Inverted microscope equipped with micromanipulation and
aspiration system.
2. Stereomicroscope with thermal plate, for maintaining materials
at 37
C.
3. Holding pipettes with a 25–30/120–150 μm inner/outer
diameter (e.g., MPH-XLG from Origio).
4. Aspiration micropipettes with a 17–10 μm inner diameter and
beveled opening (e.g., MSC-18-30 from Origio).
5. Low rim Petri dishes (as used for micromanipulation).
6. Mineral oil, suitable for mammalian embryo culture.
7. Four-well dishes.
8. 0.5 and 0.25 mL straws and 0.25 mL hemistraw (see Note 1).
Hemistraws can be prepared, under sterile conditions, using a
scalpel and 0.25 mL straw (see Fig. 2c).
9. Straw connector.
10. Labeling system for 0.5 mL straws.
11. Pipette for handling small volumes (2–10 μL), with sterile tips.
12. Stripper pipette, with stripper tips 125 μm in diameter (MXL3125, Origio).
13. Styrofoam box containing liquid nitrogen (see Note 2).
14. Fine tweezers.
15. Handling medium. PBS with no calcium or magnesium, 0.4%
(w/v) BSA and 0.1% (w/v) PVP360.
2.2 Holding Medium,
Vitrification, and
Warming Solutions
1. Holding medium (HM). Prepare by supplementing PBS with
20% fetal bovine serum (FBS) and 5 μg/mL gentamicin. Filter
and keep in the refrigerator for no longer than a week.
2. Vitrification solution 1 (VS1). Prepare in HM: 7.5% (v/v)
ethylene glycol and 7.5% (v/v) dimethyl sulfoxide (DMSO)
(see Note 3). Filter-sterilize, prepare 0.5 mL aliquots, and
freeze.
3. Vitrification solution 2 (VS2). Prepare in HM: 16.5% v/v EG,
16.5% v/v DMSO, and 0.5 M sucrose. Filter-sterilize, prepare
0.5 mL aliquots, and freeze.
4. Warming solution 1 (WS1). Dissolve in HM: 0.33 M sucrose.
Filter-sterilize, prepare 1 mL aliquots, and freeze.
5. Warming solution 2 (WS2). Dissolve in HM: 0.2 M sucrose.
Filter-sterilize, prepare 0.5 mL aliquots, and freeze.
518
Carolina Herrera