Bio-Rad Apoptosis Assays User Manual

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Homogenizer — Bio-Gen Pro200 (cat #01-01200) with a 5 mm
generator (cat #02-05075) or equivalent.

c. Dounce homogenizer — Fisher (cat #06-434) using tight pestle, or

equivalent.

Preparation of Cytosolic and Nuclear + Mitochondrial

(N + M) Fractions

Cytosolic Fraction
1. Prepare the cytosolic extraction buffer (CEB) and lysate dilution buffer

(LDB) by adding 1 tablet each of the protease inhibitors listed above
to 10 ml of each buffer. Once the tablets are in solution, store the
buffers at 4°C for up to 1 week or at –20°C for up to 3 months.

2. Pre-chill all tubes and keep samples on ice during sample

preparation. CEB additions and homogenization of samples are
summarized in Table 5.

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

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

Tissue sample with homogenizer — add 100 µl CEB to the 2 ml
screw cap tube containing tissue and mince the tissue 10–15x
using micro scissors. Add the remaining volume of CEB and

Table 5. Summary of homogenization methods for cytosolic and Nuclear +
Mitochondrial fractions.

Homogenization

Sample Type

Method

Cytosolic Extraction Buffer (CEB) Additions

Tissue: 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: <20 mg

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

(ratio of 30 µl CEB to

Pestle grinder Add 100 µl, grind, and add remaining

1 mg of tissue)

Tissue: 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 45–60 min on

5 x 10

to 2 x 10

homogenizer

ice, and dounce with ~40 passes (will depend

(750 µl total volume)

on cell line)