1.CryopreservationofMouseSpermatozoain1.8mlcryotubes.docVIP

Standard Operation Protocols (SOPs) for the Cryopreservation of Mouse Germplasm 1. Cryopreservation of Mouse Spermatozoa in 1.8ml cryotubes 2. Cryopreservation of mouse spermatozoa in French straws 3. Mouse Embryo Cryopreservation in 1,8ml cryotubes 4. Mouse Embryo Cryopreservation in 0.25 ml plastic straws 5. Superovulation protocol 6. In vitro fertilization 1. Cryopreservation of Mouse Spermatozoa in 1.8ml cryotubes Equipment Cryoprotectant agent (CPA) 18% raffinose, (Sigma; R-7630) 3% skim milk, (Difco Betalab 1878-17) made up in sterile water (Sigma; W-1503). 1.8ml Nunc cryotubes. Cryotube rack. Micro centrifuge and 1.5ml microfuge tubes. 35mm culture dishes, (e.g. Falcon 3001). Deep polystyrene box with lid (suitable for holding liquid nitrogen). Small dewar for liquid nitrogen. CO2 incubator (37°C). Hot block held at 37°C. Sexually mature male mice at least 8 weeks old (not recently mated). Method Place 9ml Sigma H2O (W-1503) in a screw top 15ml Falcon tube (2097) and equilibrate to 60oC in a water bath. Add 1.8g raffinose and dissolve by gentle inversion. Add 0.3g skim milk and dissolve by gentle inversion. Make up to 10ml if necessary. Aliquot into microfuge tubes and centrifuge at 14,000 rpm for 10 min. Collect supernatant and filter the solution using a 0.45μm syringe end filter. Place 1.1ml aliquots into cryotubes and store at -20oC. Cryopreservation method To prepare the cooling apparatus place a platform (e.g. the insert from a Gilson yellow tip box) into the polystyrene box. This acts as a support for the cryotube rack. Carefully pour liquid nitrogen into the polystyrene box to just cover the platform. Place a cryotube rack on top of the platform so that it is suspended in liquid nitrogen vapour. Replace the lid on the polystyrene box and allow it to fill with vapour. Replenish the liquid nitrogen as necessary during the freezing session, but do not allow the level to rise above the platform. Thaw one 1.1ml aliquot of cryoprotectant solution fo