3.2 Preparation
of FHM Medium
FHM medium is modified KSOM [25] where part of the bicarbonate is replaced with HEPES buffer. It is often used for washing and
handling mouse embryos on bench, not in the CO 2 incubators.
FHM can be used for post-thaw dilution to measure sperm motility
on the bench.
1. Bring an appropriate size Griffin beaker, pour embryo tested
Milli-Q water (~80% of the total volume of media), and then
add the components in gram as listed in Table 2. 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 completely 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 2).
3. Add 4.0 g/1 L bovine serum albumin fraction V (Sigma
A7638) into the stock solution, and let it dissolve slowly (see
Note 3).
Table 2
Composition of stock solutions used to make FHM medium
Components
Company
Add per L
Concentration (mM)
CaCl 2 .2H 2 O
Sigma C7902
0.251 g
1.71
KCl
Sigma P5405
0.1864 g
2.5
NaCl
Sigma S5886
5.5518 g
95
NaHCO 3
Sigma S5761
0.42 g
4.0
D-glucose
Sigma G6152
0.036 g
0.2
Sodium lactate (60% syrup)
Sigma L7900
1.740 mL
11.77
Sodium pyruvate
Sigma P4562
0.022 g
0.2
Penicillin G potassium salt
Sigma P7794
0.06 g
N/a
Streptomycin sulfate
Sigma S1277
0.0125 g
N/a
KH 2 PO 4
Sigma P5655
0.0476 g
0.35
MgSO 4 .7H 2 O
Sigma M1880
0.0493 g
0.2
EDTA
Sigma E6635
0.0037 g
0.01
HEPES
Sigma H6147
4.7662 g
20
Phenol red (0.5%)
Sigma P0290
0.66 mL
N/a
GlutaMAX 1
Invitrogen 35050-061
5 mL
1.0
MEM EAA 50Â
Gibco 11130-051
10 mL
N/a
NE amino acids
Gibco 11140-050
5 mL
N/a
Mouse Sperm Cryopreservation
405
of FHM Medium
FHM medium is modified KSOM [25] where part of the bicarbonate is replaced with HEPES buffer. It is often used for washing and
handling mouse embryos on bench, not in the CO 2 incubators.
FHM can be used for post-thaw dilution to measure sperm motility
on the bench.
1. Bring an appropriate size Griffin beaker, pour embryo tested
Milli-Q water (~80% of the total volume of media), and then
add the components in gram as listed in Table 2. 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 completely 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 2).
3. Add 4.0 g/1 L bovine serum albumin fraction V (Sigma
A7638) into the stock solution, and let it dissolve slowly (see
Note 3).
Table 2
Composition of stock solutions used to make FHM medium
Components
Company
Add per L
Concentration (mM)
CaCl 2 .2H 2 O
Sigma C7902
0.251 g
1.71
KCl
Sigma P5405
0.1864 g
2.5
NaCl
Sigma S5886
5.5518 g
95
NaHCO 3
Sigma S5761
0.42 g
4.0
D-glucose
Sigma G6152
0.036 g
0.2
Sodium lactate (60% syrup)
Sigma L7900
1.740 mL
11.77
Sodium pyruvate
Sigma P4562
0.022 g
0.2
Penicillin G potassium salt
Sigma P7794
0.06 g
N/a
Streptomycin sulfate
Sigma S1277
0.0125 g
N/a
KH 2 PO 4
Sigma P5655
0.0476 g
0.35
MgSO 4 .7H 2 O
Sigma M1880
0.0493 g
0.2
EDTA
Sigma E6635
0.0037 g
0.01
HEPES
Sigma H6147
4.7662 g
20
Phenol red (0.5%)
Sigma P0290
0.66 mL
N/a
GlutaMAX 1
Invitrogen 35050-061
5 mL
1.0
MEM EAA 50Â
Gibco 11130-051
10 mL
N/a
NE amino acids
Gibco 11140-050
5 mL
N/a
Mouse Sperm Cryopreservation
405