5. Filter into an appropriately sized sterile bottle using 0.22 μm
pore size filters.
6. Aliquot into desired volume, and label the tubes with make and
expiration date (3 months from make date), initials, and batch
number.
7. Store in À20
C freezer for up to 3 months.
3.4 Preparation
of HTF Medium
Human tubal fluid (HTF) is a bicarbonate-buffered medium which
was formulated to perform IVF in mice using incubator having 5%
CO 2 and 95% humidified air at 37
C [26].
1. Bring an appropriate size Griffin beaker, pour embryo transfer
water (~80% of the total volume), and then add the following
components in gram as depicted in Table 4. All the recipes are
calculated based on 1 L final volume, so adjust the volume of
the media for your needs (see Note 1).
2. After all components are dissolved, transfer the contents to the
appropriate volumetric flask and bring to desired volume by
rinsing out the Griffin beaker with embryo tested Milli-Q water
and adding it to the volumetric flask (see Note 4).
3. Add 4.0 g/1 L bovine serum albumin fraction V, fatty acid free
(EMD Millipore 126575) on top of the HTF stock solution,
and let it dissolve completely (see Note 3).
4. Gas the media with 5% CO 2 /nitrogen balance for ~30 min.
Table 4
Composition of in vitro fertilization human tubal fluid (HTF)
Components
Company
Add per L
Concentration (mM)
CaCl 2
Sigma C5670
0.570 g
5.14
KCl
Sigma P5405
0.350 g
4.69
NaCl
Sigma S5886
5.938 g
101.6
NaHCO 3
Sigma S5761
2.100 g
25
D-glucose
Sigma G6152
0.500 g
2.78
Sodium lactate (60% syrup)
Sigma L7900
3.400 mL
21.4
Sodium pyruvate
Sigma P4562
0.037 g
0.33
Penicillin G potassium salt
Sigma P7794
0.075 g
N/a
Streptomycin sulfate
Sigma S1277
0.05 g
N/a
KH 2 PO 4
Sigma P5655
0.054 g
0.37
MgSO 4 .7H 2 O
Sigma M1880
0.0490 g
0.20
Phenol red (0.5%)
Sigma P8136
0.4 mL
N/a
Mouse Sperm Cryopreservation
407
pore size filters.
6. Aliquot into desired volume, and label the tubes with make and
expiration date (3 months from make date), initials, and batch
number.
7. Store in À20
C freezer for up to 3 months.
3.4 Preparation
of HTF Medium
Human tubal fluid (HTF) is a bicarbonate-buffered medium which
was formulated to perform IVF in mice using incubator having 5%
CO 2 and 95% humidified air at 37
C [26].
1. Bring an appropriate size Griffin beaker, pour embryo transfer
water (~80% of the total volume), and then add the following
components in gram as depicted in Table 4. All the recipes are
calculated based on 1 L final volume, so adjust the volume of
the media for your needs (see Note 1).
2. After all components are dissolved, transfer the contents to the
appropriate volumetric flask and bring to desired volume by
rinsing out the Griffin beaker with embryo tested Milli-Q water
and adding it to the volumetric flask (see Note 4).
3. Add 4.0 g/1 L bovine serum albumin fraction V, fatty acid free
(EMD Millipore 126575) on top of the HTF stock solution,
and let it dissolve completely (see Note 3).
4. Gas the media with 5% CO 2 /nitrogen balance for ~30 min.
Table 4
Composition of in vitro fertilization human tubal fluid (HTF)
Components
Company
Add per L
Concentration (mM)
CaCl 2
Sigma C5670
0.570 g
5.14
KCl
Sigma P5405
0.350 g
4.69
NaCl
Sigma S5886
5.938 g
101.6
NaHCO 3
Sigma S5761
2.100 g
25
D-glucose
Sigma G6152
0.500 g
2.78
Sodium lactate (60% syrup)
Sigma L7900
3.400 mL
21.4
Sodium pyruvate
Sigma P4562
0.037 g
0.33
Penicillin G potassium salt
Sigma P7794
0.075 g
N/a
Streptomycin sulfate
Sigma S1277
0.05 g
N/a
KH 2 PO 4
Sigma P5655
0.054 g
0.37
MgSO 4 .7H 2 O
Sigma M1880
0.0490 g
0.20
Phenol red (0.5%)
Sigma P8136
0.4 mL
N/a
Mouse Sperm Cryopreservation
407