9. Transfer the straw to a liquid nitrogen storage vessel (see
Note 12).
10. Prior to warming the straw, place 1 mL of 1 M sucrose solution
in a culture dish, half fill a 1 mL syringe with 1 M sucrose, and
place 2Â 50 μL droplets of sucrose solution and 2Â 50 μL
droplets of PBS with protein supplement in a culture dish.
Label the droplets.
11. Wearing suitable protective equipment, remove the straw from
liquid nitrogen storage. Hold the straw in air for 10 s, hold
either end of the straw not the area containing the oocytes, and
then plunge the straw into the water bath at 20
C for 10 s (see
Note 16).
12. Wipe the straw dry and cut through the straw using a scalpel in
the area containing the sucrose. Remove the plug from the
other end, if used, or cut with a scalpel. Attach the syringe
containing sucrose to one end of the straw and hold the other
end over the dish containing 1 mL 1 M sucrose solution. Flush
the contents of the straw and syringe into the dish, and ensure
good mixing.
13. Immediately begin to look for the oocytes using the dissecting
microscope (see Note 17). As soon as the oocytes are identified
place them in one of the droplets of 1 M sucrose solution.
14. Immediately move the oocytes into the second droplet of 1 M
sucrose solution. When the oocytes have been in contact with
1 M sucrose for a total of 5 min, transfer the oocytes into a
droplet of PBS, and leave for 10 min at room temperature.
Move the oocytes into the second droplet of PBS and leave
them for 10 min on the hotplate.
15. The oocytes should then be placed in oocyte culture medium,
within an incubator, (2–3 h for human, 30 min for mouse)
prior to addition of sperm (see Note 13).
4 Notes
1. Ice nucleation can be initiated automatically by some freezing
machines or can be initiated manually by removing one end
of the straws from the machine and touching the solution with
forceps or cotton tipped applicators/cotton swabs that have
been pre-cooled in liquid nitrogen or by insertion of an ice
crystal from a pipette tip.
2. Straws can be sealed by heating of the ends (and is required for
clinical samples), although care must be taken not to heat the
solution containing the oocytes, or by inserting plugs, which
should be wet in order to give a good seal, or with sealing
powder.
448
Victoria Keros and Barry J. Fuller
Note 12).
10. Prior to warming the straw, place 1 mL of 1 M sucrose solution
in a culture dish, half fill a 1 mL syringe with 1 M sucrose, and
place 2Â 50 μL droplets of sucrose solution and 2Â 50 μL
droplets of PBS with protein supplement in a culture dish.
Label the droplets.
11. Wearing suitable protective equipment, remove the straw from
liquid nitrogen storage. Hold the straw in air for 10 s, hold
either end of the straw not the area containing the oocytes, and
then plunge the straw into the water bath at 20
C for 10 s (see
Note 16).
12. Wipe the straw dry and cut through the straw using a scalpel in
the area containing the sucrose. Remove the plug from the
other end, if used, or cut with a scalpel. Attach the syringe
containing sucrose to one end of the straw and hold the other
end over the dish containing 1 mL 1 M sucrose solution. Flush
the contents of the straw and syringe into the dish, and ensure
good mixing.
13. Immediately begin to look for the oocytes using the dissecting
microscope (see Note 17). As soon as the oocytes are identified
place them in one of the droplets of 1 M sucrose solution.
14. Immediately move the oocytes into the second droplet of 1 M
sucrose solution. When the oocytes have been in contact with
1 M sucrose for a total of 5 min, transfer the oocytes into a
droplet of PBS, and leave for 10 min at room temperature.
Move the oocytes into the second droplet of PBS and leave
them for 10 min on the hotplate.
15. The oocytes should then be placed in oocyte culture medium,
within an incubator, (2–3 h for human, 30 min for mouse)
prior to addition of sperm (see Note 13).
4 Notes
1. Ice nucleation can be initiated automatically by some freezing
machines or can be initiated manually by removing one end
of the straws from the machine and touching the solution with
forceps or cotton tipped applicators/cotton swabs that have
been pre-cooled in liquid nitrogen or by insertion of an ice
crystal from a pipette tip.
2. Straws can be sealed by heating of the ends (and is required for
clinical samples), although care must be taken not to heat the
solution containing the oocytes, or by inserting plugs, which
should be wet in order to give a good seal, or with sealing
powder.
448
Victoria Keros and Barry J. Fuller