Bio-Rad Apoptosis Assays User Manual

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2. Prechill all tubes and keep samples on ice during sample preparation.

LDB additions and homogenization of samples are summarized in
Table 6.

3. Homogenize the samples using the following guidelines (depending on

sample type, additional time may be needed to ensure the sample is
completely homogenized).

a. Tissue sample with homogenizer — add 100 µl LDB to the 2 ml

screw cap tube containing tissue and mince the tissue 10–15x using
micro scissors. Add the remaining volume of LDB and 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 LDB 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 LDB, and remove any large tissue fragments
that did not homogenize. Incubate the processed samples on ice
for 10 min.

Table 6. Summary of homogenization methods for total extracts.

Homogenization

Sample Type

Method

Lysate Dilution Buffer (LDB) Additions

Tissue sample: 20–30 mg

Homogenizer Add 100 µl, mince, add 650 µl, and homogenize

(750 µl total volume)

Pestle grinder Add 100 µl, grind, and add 650 µl

Tissue sample: <20 mg

Homogenizer Add 100 µl, mince, add remaining, and homogenize

(ratio of 30 µl LDB to

Pestle grinder Add 100 µl, grind, and add remaining

1 mg of tissue)

Tissue sample: fine needle Homogenizer Add 100 µl, mince, add 250 µl, and homogenize
aspirate (350 µl total volume) Pestle grinder Add 100 µl, grind, and add 250 µl

Cultured cells:

Dounce

Resuspend in 750 µl, incubate 30 min on ice,

5 x 10

to 2 x 10

homogenizer

and dounce with ~40 passes (depends on

(750 µl total volume)

cell line)