Bring all medias to 37 C temperature.

Care fully Layer 1 ml of 40% (upper layer) on 80% (lower layer) and load the liquefied semen sample on top of the upper layer as shown in the picture. Centrifuge at 2000 rpm for 15 mins

Notes:

1. Use well liquefied semen samples only

2. Use 1 to 1.5 ml of the semen sample

After Centrifugation discard the supernatant with out disturbing the pellet

Notes:

1. Pellet is nothing but concentrated sperms

2. Disturbing the pellet may lead to loss of sperms

Add 2ml of Sperm Wash media to the pellet and mix well. Transfer the contents to a fresh tube and Centrifuge at 1600 rpm for 8 mins.

Notes:

1. This is wash step 1

2. Wash step removes silica particles from the density gradient media

3. repeat the wash step for thorough washing of sperms

Discard the supernatant of the Wash Step 1

Notes:

1. Pellet is nothing but concentrated sperms

2. Disturbing the pellet may lead to loss of sperms

Add 2ml of Sperm Wash media to the pellet and mix well. Centrifuge again at 1600 rpm for 8 mins.

Notes:

1. This is wash step 2

2. Wash step removes silica particles from the density gradient media

Discard 1.5ml of supernatant from the Wash Step 2

Notes:

1. Pellet is nothing but concentrated sperms

2. Disturbing the pellet may lead to loss of sperms

Add rest of the 0.5 ml of Sperm Wash media to the pellet in a very gentle manner without disturbing the pellet. Leave the tube at 45 degrees inclination for the incubation at 37 C temperature for 30 to 45 mins

Notes:

1. During the swim-up sperms from pellet reach the top fraction of the media

2. After the incubation remove the top fraction and keep at 37 C until use.