Bio-Rad Apoptosis Assays User Manual

Page 17

homogenize the minced tissue for approximately 5 sec on medium
power. Homogenized tissue samples should be free of large tissue
fragments. If large fragments are visible in the sample, homogenize
for an additional 5 sec. Any large fragments remaining after the
additional 5 sec homogenization step should be removed before
proceeding. Incubate processed samples on ice for 10 min.
After incubation, transfer the sample to a prechilled 1.5 ml
microcentrifuge tube.

b. Tissue sample with pestle grinder — add 100 µl CEB to the

1.5 ml pestle tube containing tissue sample. Grind the tissue for
approximately 10 sec or until no large pieces remain. Then add the
remaining volume of CEB, and remove any large fragments that did
not homogenize. Incubate processed samples on ice for 10 min.

Cultured Cells

i. Collect the cells (5 x 10

to 2 x 10

) by centrifugation at 4°C for

5 min at 500 x g

ii. Wash the cells in 1–2 ml of ice-cold PBS. Centrifuge at 4°C for

5 min at 500 x g. Remove supernatant.

iii. Freeze the pellet at –80°C for a minimum of 15 min. Remove and

place on ice.

iv. Add 750 µl CEB to the pellet, resuspend the cells by pipetting up

and down 10–15x, and vortex for 5 sec.

v. Incubate the suspension on ice for 45–60 min.

vi. Homogenize on ice using approximately 40 passes with a Dounce

homogenizer.

Note:

The number of passes will depend on the cell line and may

need to be adjusted by the end user for effective lysis.

vii. Transfer the homogenate to a 1.5 ml microcentrifuge tube.

4. Centrifuge the homogenized sample at 4°C for 10 min at 10,000 x g.

5. Carefully remove the supernatant with a pipet and transfer to a pre-

chilled 1.5 ml microcentrifuge tube. Measure the volume of cytosolic
fraction removed, add an equal amount of LDB, and vortex. Label as
cytosolic fraction.