30
3 Methods
1. Sacrifice a mouse sticking to the rules constructed by your animal care committee.
2. Remove the brain, heart and liver, and be sure that the organs
are clean of hair and other wastes. Olfactory bulbs of brain are
removed and the brain is divided into two hemi brains.
3. The sacrificed organs are washed with sterile and cold PBS,
transferred to separate tubes containing 15 mL sterile PBS and
then ultrasonicated for 3 min under 750 W power and 20 kHz
frequency ultrasonication conditions (Sonics VCX 750) in
order to dissociate the tissue and release the extracellular vesicles (see Notes 1 and 2).
4. Centrifuge the tissue homogenates at 300 × g, 2000 × g and
13,000 × g for 15 min at 4 °C, respectively, and remove the cellular debris, microvesicles and apoptotic bodies.
5. Transfer 10 mL of the supernatant into 50 mL tubes and add
10 mL 20% PEG 4000 solution.
6. Incubate the tissue homogenate-PEG mixture overnight at
4 °C.
7. Centrifuge the mixture at 13,000 × g at 4 °C and discard the
supernatant without disturbing the exosome pellet.
8. Resuspend the pellet in 100 μL PBS kept at 4 °C and store at
−20 °C for further use.
Quantification of the exosomal protein content is performed following the manufacturer’s instruction. Briefly:
1. Exosomes are diluted 1:2 in PBS.
2. Exosome lysate is obtained by adding an equal volume of RIPA
buffer including protease inhibitors.
3. Pipette 25 μL of each bovine serum albumin (BSA) standard
with concentrations between 12.5 μg.mL
−1
and 2.5 mg.mL
−1
and organ-derived exosome samples into a 96-well microplate.
The procedure is carried out in triplicate.
4. Add 200 μL of the working reagent into each well and shake
the plate on a platform shaker for 30 s.
5. Cover the plate and incubate at 37 °C for 30 min.
6. Measure the absorbance at 562 nm on a spectrophotometer
(Perkin Elmer 1420 Multilabel Counter).
Determination of exosome particle count is performed following
the manufacturer’s instruction of CD63 ExoElisa Kit (System
Bioscience). Briefly:
3.1 Isolation
of Exosomes
from Brain, Heart,
and Liver
3.2 Protein
Quantification
of Exosomes Using
BCA Test
3.3 Exosome Particle
Counting with CD63
ELISA Kit
Burak Derkus and Emel Emregul
3 Methods
1. Sacrifice a mouse sticking to the rules constructed by your animal care committee.
2. Remove the brain, heart and liver, and be sure that the organs
are clean of hair and other wastes. Olfactory bulbs of brain are
removed and the brain is divided into two hemi brains.
3. The sacrificed organs are washed with sterile and cold PBS,
transferred to separate tubes containing 15 mL sterile PBS and
then ultrasonicated for 3 min under 750 W power and 20 kHz
frequency ultrasonication conditions (Sonics VCX 750) in
order to dissociate the tissue and release the extracellular vesicles (see Notes 1 and 2).
4. Centrifuge the tissue homogenates at 300 × g, 2000 × g and
13,000 × g for 15 min at 4 °C, respectively, and remove the cellular debris, microvesicles and apoptotic bodies.
5. Transfer 10 mL of the supernatant into 50 mL tubes and add
10 mL 20% PEG 4000 solution.
6. Incubate the tissue homogenate-PEG mixture overnight at
4 °C.
7. Centrifuge the mixture at 13,000 × g at 4 °C and discard the
supernatant without disturbing the exosome pellet.
8. Resuspend the pellet in 100 μL PBS kept at 4 °C and store at
−20 °C for further use.
Quantification of the exosomal protein content is performed following the manufacturer’s instruction. Briefly:
1. Exosomes are diluted 1:2 in PBS.
2. Exosome lysate is obtained by adding an equal volume of RIPA
buffer including protease inhibitors.
3. Pipette 25 μL of each bovine serum albumin (BSA) standard
with concentrations between 12.5 μg.mL
−1
and 2.5 mg.mL
−1
and organ-derived exosome samples into a 96-well microplate.
The procedure is carried out in triplicate.
4. Add 200 μL of the working reagent into each well and shake
the plate on a platform shaker for 30 s.
5. Cover the plate and incubate at 37 °C for 30 min.
6. Measure the absorbance at 562 nm on a spectrophotometer
(Perkin Elmer 1420 Multilabel Counter).
Determination of exosome particle count is performed following
the manufacturer’s instruction of CD63 ExoElisa Kit (System
Bioscience). Briefly:
3.1 Isolation
of Exosomes
from Brain, Heart,
and Liver
3.2 Protein
Quantification
of Exosomes Using
BCA Test
3.3 Exosome Particle
Counting with CD63
ELISA Kit
Burak Derkus and Emel Emregul