GENERATION OF LYSATE INOCULATION MATERIALS

Acquire the tissue from 4-month-old asymptomatic TgA53T (Line G2-3) and end-stage tissue harvest and store at -80°C prior to use.

Weigh the tissue and suspend it in 0.9% sterile saline (1:10 w/vol).

Homogenize and centrifuge for 0h 5m 0s at 3000x g,0h 0m 0s at 4°C. The supernatant (S3000) is obtained.

Centrifuge S3000 for 0h 45m 0s at 150000x g,0h 0m 0s at 4°C. The supernatant (S150) – Highly soluble fraction is obtained.

Wash pellet and resuspend in sterile saline (half of original volume) by sonication (3 x 10 min pulses). Resuspended pellet (P150) – Insoluble fraction is obtained.

Wash pellet and resuspend in sterile saline (half of original volume) by sonication for 0h 0m 10s pulses) (1/3).

Wash pellet and resuspend in sterile saline (half of original volume) by sonication for 0h 0m 10s pulses) (2/3).

Wash pellet and resuspend in sterile saline (half of original volume) by sonication for 0h 0m 10s pulses) (3/3).

Homogenize freshly harvested tissues (1:10 w/vol) in lysis buffer (refer material section).

Centrifuge at 1000x g,0h 0m 0s and collect the supernatant.

Centrifuge at 10000x g,0h 0m 0s. The pellet containing mitochondria is obtained.

Centrifuge supernatant at 100000x g,0h 0m 0s. The supernatant containing cytosol and pellet containing ER are obtained.