Collection and Washing of Sperm from the AcroSensE Mouse Model for Real-Time Imaging

Begin by collecting sperm from the cota epididymus, isolated from a transgenic model mouse expressing AcroSensE. Perform the sperm collection in a 3.5 centimeter tissue culture dish containing 0.5 milliliters of MW medium by allowing a 15 minute swim out procedure at 37 degrees Celsius. Then using fine forceps, carefully remove any epididymal tissue from the swim out collection dish.

Transfer the medium containing the sperm into a 15 milliliter conical tube and using a swinging bucket rotor centrifuge the suspension at 100 G for one minute at room temperature. With the help of a plastic pipette, collect the MW supernatant containing the sperm while discarding any pellet of gross tissue debris and transfer it to a round bottom tube. Adjust the supernatant volume to one milliliter by adding 500 microliters of MW medium prewarmed to 37 degrees Celsius.

Centrifuge this mixture at 400 G for eight minutes at room temperature in a round bottom tube. Then carefully remove the supernatant while leaving behind the loosely pelleted sperm. Using a large bore transfer pipe, re-suspend the sperm by gently tapping the tube manually known as finger flicking.

Once the sperm are evenly re-suspended, transfer them into a new round bottom tube.