29. Appropriate-sized Griffin beaker and volumetric flask, stir bar,
weigh paper, and spatula.
30. 0.22 μm and 0.45 μm pore size filters.
3 Methods
3.1 Preparation
of Mouse Sperm
Freezing Solution
Components of the mouse sperm freezing solution are presented in
Table 1.
1. Bring an appropriate size Griffin beaker, and add 75 mL warm
embryo transfer water (Sigma W1503) at 60
C.
2. Slowly add raffinose and use stir bar until raffinose is completely
dissolved.
3. Slowly add dehydrated skim milk, and stir the solution until all
skim milk particles disappear.
4. Add monothioglycerol into solution. Pipette slowly as it is
viscous.
5. Add L-glutamine into solution and let it completely dissolve.
6. Bring a final volume of 100 mL by rinsing out the Griffin
beaker with embryo transfer water into a volumetric flask.
7. Equally aliquot the solution into two sterile 50 mL roundbottom centrifuge tubes.
8. Centrifuge for 1 h at 18,000 Â g at 4
C until the supernatant
becomes clear.
9. Carefully pipette off the supernatant into a graduated cylinder
without disturbing the pellet.
10. Filter the supernatant through a 0.22 μm pore size filter
(if needed, prefilter using 0.45 μm pore size).
11. The medium should have an osmolality of ~520 mOsm/kg.
12. The freezing solution can be stored at À20
C for up to
1 month.
Table 1
Preparation of mouse sperm cryopreservation solution
Components
Company
Amount
(D+) raffinose pentahydrate
Sigma R-0514
18 g
Nonfat dehydrated skim milk
Fisher 232100
3 g
Monothioglycerol
Sigma M6145
4 μl
L-glutamine
Sigma G8540
1.460 g
Final volume embryo transfer water
Sigma W1503
100 mL
404
Yuksel Agca and Cansu Agca
weigh paper, and spatula.
30. 0.22 μm and 0.45 μm pore size filters.
3 Methods
3.1 Preparation
of Mouse Sperm
Freezing Solution
Components of the mouse sperm freezing solution are presented in
Table 1.
1. Bring an appropriate size Griffin beaker, and add 75 mL warm
embryo transfer water (Sigma W1503) at 60
C.
2. Slowly add raffinose and use stir bar until raffinose is completely
dissolved.
3. Slowly add dehydrated skim milk, and stir the solution until all
skim milk particles disappear.
4. Add monothioglycerol into solution. Pipette slowly as it is
viscous.
5. Add L-glutamine into solution and let it completely dissolve.
6. Bring a final volume of 100 mL by rinsing out the Griffin
beaker with embryo transfer water into a volumetric flask.
7. Equally aliquot the solution into two sterile 50 mL roundbottom centrifuge tubes.
8. Centrifuge for 1 h at 18,000 Â g at 4
C until the supernatant
becomes clear.
9. Carefully pipette off the supernatant into a graduated cylinder
without disturbing the pellet.
10. Filter the supernatant through a 0.22 μm pore size filter
(if needed, prefilter using 0.45 μm pore size).
11. The medium should have an osmolality of ~520 mOsm/kg.
12. The freezing solution can be stored at À20
C for up to
1 month.
Table 1
Preparation of mouse sperm cryopreservation solution
Components
Company
Amount
(D+) raffinose pentahydrate
Sigma R-0514
18 g
Nonfat dehydrated skim milk
Fisher 232100
3 g
Monothioglycerol
Sigma M6145
4 μl
L-glutamine
Sigma G8540
1.460 g
Final volume embryo transfer water
Sigma W1503
100 mL
404
Yuksel Agca and Cansu Agca