Mouse husbandry and tissue collection

Cauda sperm collection protocol

1. Dissect out cauda epididymis from an adult mouse and transfer to a 35mm dish containing 1ml of pre-warmed Whitten’s Media (100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM Glucose, 1 mM Pyruvic acid, 4.8 mM Lactic acid (hemicalcium), and HEPES 20 mM).
2.  Gently squeeze the epididymis to allow the caudal fluid to ooze out in the dish. 
3.  Incubate at 37°C for 15 minutes
4.  After incubation transfer the sperm containing media to a fresh tube and incubate for another 15 minutes
5.  Collect the supernatant, which contains the motile sperm and epididymosome. 

If epididymis tissue is needed, then wash it with PBS in the plate 3 times, followed by transferring to 1.5 ml tube and flash freeze. 

1.  Centrifuge supernatant at 3000 X g for 3 minutes at room temperature. This supernatant contains epididymosome. Transfer it to another tube and continue from step 14 for epididymosome isolation.
2.  Wash sperm pellet with 1X PBS at 3000g 5 min
3.  Centrifuge at 3000 X g for 5minutes at room temperature
4.  Add 500ul somatic lysis buffer (0.01% SDS and 0.005% Triton-X in water) and incubate on ice for 10 minutes 

10. Centrifuge at 5000 X g for 5 minutes at 4C

11. Wash with 1X PBS at 5000g 5 min at 4C

13. Flash freeze the sperm pellets at -80°C

Whitten’s medium 500 ml:

100mM NaCl:                  33.3 ml 1.5 M NaCl

4.7 mM KCl:                     2.3 ml 1 M KCl

1.2 mM KH2PO4:           0.6 ml 1 M KH2PO4

1.2 mM MgSO4:              0.6 ml 1 M MgSO4

5.5 mM Glucose:            495 mg Glucose

1 mM Pyruvic acid:       35ul (Pyruvic acid Sigma 107360-100G)

4.8 mM Lactic acid:        523.2 mg (Calcium L-lactate hydrate)

20 mM HEPES:              100 ml 100 mM HEPES

Add water to                  500ml then filter it, and stored at 4C