GBE 302 LABORATORY

GBE 302
BIOCHEMISTRY-II LABORATORY

EXPERIMENT 4
MITOCHONDRIA ISOLATION

SUBMITTED BY

Bilge Deniz Yilmaz

COURSE INSTRUCTER: Prof. Dr. Dilek Telci

SUBMITTED TO:

Ayca Ece Nezir

Ipek Bedir
Özge Deniz Yeşil

Experiment Date: 18.03.21

Submission Date: 31.03.21

Yeditepe University

Genetics and Bioengineering Department

Istanbul
 1. OBJECTIVE:

The purpose of this experiment is to isolate mitochondria from fresh mouse liver tissue
through differential centrifugation technique by using sucrose, HEPES and EDTA containing
homogenization and suspension buffers and at the end the yield of mitochondria isolation
was calculated.

2. INTRODUCTION:

According to endosymbiotic theory mitochondria is present like a cell itself inside the main cell,
it has two therefore unlike other compartments of the cell it can produce its own proteins.
Functions as the main source of energy inside the cell, reactive oxygen species signaling,
regulates the membrane potential and also cellular metabolism, calcium signaling and apoptosis
programmed cell death. The amount of mitochondria that a tissue contains in its structure varies
accrordingly to the amount of energy it needs. Liver, muscle, kidney and brain cells contains
significantly higher amount of mitochondria than other organs with respect to their metabolic
activities [1].

Apoptosis is programmed cell death occurs in two distinct pathway as extrinsic and intrinsic.
Intrinsic pathway can be triggered by several factors such as oxidative stress in metabolic
pathway or endoplasmic reticulum, hypoxia or when the cellular DNA is damaged and cannot be
repaired. It plays an important role in elimination of damaged cells and development [2,3]

The working mechanism of Janus Green-B relies on oxidation of sugar during cellular
respiration.
3. APPARATUS:

● 0.1 grams of fresh mouse liver

● 0.25 M sucrose in 10 mM HEPES at pH 7.5 (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) as homogenization buffer
● 0.25 sucrose dissolved in 10 mM HEPES (at pH 7.5) 1 mM EDTA as suspension
buffer
● Teflon homogenizer
● bistoury
● Janus Green-B
● Hemocytometer
● microscope
● Refrigerated Centrifuge

4. PROCEDURE:

Removed liver organ was cut into small pieces and weighted as being 0.1 grams. Isolated
liver tissue was washed with PBS and cut into small pieces. Homogenization buffer was
preapared by dissolving 9 ml of 0.25 mM concentrated sucrose solution in 10 mM HEPES
buffer that is adjusted at pH 7.5 and added onto liver pieces. Buffer treated tissues were
ruptured mechanically by Teflon homogenizer. Homogenized sample was centrifuged at
16.000 G, 40C for 5 minutes. Supernatant was discarded and 1 ml of suspension buffer was
added into pellet. Pellet was dissolved through gentle pipetting. 100 ul of isolated cell sample
was mixed with 100 ul of Janus Green reagent (as being 1:1 ratio) and transferred in
hemocytometer and the number of Janus Green-B taken mitochondria was counted under 4X
objective. Results were recorded and cell concentration was calculated.

4.1 CALCULATIONS:
● 9 ml of 0.25 M sucrose + 10 mM HEPES buffer was used for each gram of
tissue according to the literature and experimental protocol therefore the
 calculation of required amount of buffer for 0.1 grams of tissue can be
calculated as;
For 1 g of liver = 9 ml buffer
1.1 gram of liver = 0.9 ml of sucrose solution in HEPES buffer should be used.

● Calculation of the total number of isolated mitochondria inside the mouse

liver tissue;
Dilution factor: 1 mm x 1mm x 0.1 mm= 0.1 mm3 or 104 ml
At the end of the experiment 30 mitochondria was counted on hemocytometer
and according to equation it is calculated as;

Total # of isolated mitochondria from the liver tissue = 30 x 104 x 1 ml = 300.000

mitochondria/ml

5. RESULTS
At the end of the mitochondria isolation from liver tissue process, 30 mitochondria were
counted by using hemocytometer and the yield of isolated mitochondria was calculated
by comparing the experimental results with the literature as following;

Actual yield / Experimental yield x100 = %yield

Theoretical yield of mitochondria isolation from rat brain = 10 mg which is not a comparable
result in terms of unit difference.
 6. DISCUSSION:

In this experiment, it was aimed to isolate mitochondria organelle and it was proceeded by using
mouse liver tissue. In this manner brain, hearth and muscle tissues can be used as well in terms
of the high amount of mitochondria that they contain in their structure. The choice of which
tissue will be used for the isolation process is determined according to the purpose of the
experiment. The liver is softer when it is compared to hearth and muscle and also more
homogenized structured organ than the brain therefore it is easier to isolate mitochondria from
the liver. In order to test the freshness of the isolated liver tissue, it can be treated with hydrogen
peroxide; when the catalase enzyme inside the liver comes in contact with its substrate, it
converts it to water and oxygen which will appear as bubbles. Janus Green-B reagent changes its
color to blue when it is oxidized in other words it allows us to measure the presence and amount
of oxygen of the sample both qualitatively and quantitatively. It is also a membrane permeable
reagent therefore working with freshly isolated tissue is important.
In an isolation procedure it is critical to stabilizing the physiological pH of the cell culture, in this
manner HEPES buffer, which is a zwitterionic sulfonic acid buffer that comes with high
solubility and membrane impermeability features, was used in this experiment and according to
the literature it is more likely used than MOPS (3-(N-morpholino) propane sulfonic acid),
HEPBS(N-(2-Hydroxyethyl)piperazine-N'-(4-butane sulfonic acid)) can also be used. HEPES
buffer is more preferable than Tris buffer because HEPES works at low temperatures but Tris pH
effected by lowering temperatures. Since the experiment is performed on ice, preventing the
degradation of cell and proteins, HEPES is preferred.

[6]. 
The purpose of adding EDTA (ethylene diamine tetraacetic acid) is to increase the activity of
lysis buffer for its chelating feature. Generally, lysis buffers contain chelating agents such as
EDTA or EGTA and the general reason is these agents reduces the activity of protease or DNase,
enzymes that require magnesium ions to function. During the tissue fractionation and isolation,
the Calcium(Ca+2) and Magnesium (Mg+2) ions are thrown out of the mitochondrial matrix into
the solution which will reduce the effect of lysis buffer. the electron micrographic studies show
that the presence of EDTA itself leads to condensation and dehydration of the mitochondrial
matrix and also it is necessary for the removal of a significant amount of water from the inner
membranous spaces. Furthermore, when the energy is absent, the EDTA agent reduces the
permeability of mitochondrial membranes, causes the water to be trapped in between two
semipermeable membranes, and results in dehydration of the matrix [7].
 The use of sucrose buffer in a cell or organelle isolation procedure increases the yield of
obtained pellet. Because of the molecular size of sucrose and the density, the buffer does not
interact with the cellular components thus it prevents any risk of harm during centrifugation in
other words it reduces the effect of shear stress on cells. It is important to maintain the isotonic
state of a buffer to prevent premature lysis of mitochondrial membrane and it is maintained at
0.25 M concentrated sucrose buffer[8].

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[6] Lepe-Zuniga, J., Zigler, J., & Gery, I. (1987). Toxicity of light-exposed Hepes
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[7] LYNN, W. S., Jr, FORTNEY, S., & BROWN, R. H. (1964). ROLE OF EDTA AND
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[8] Bustamante, E., Soper, J. W., & Pedersen, P. L. (1977). A high-yield preparative
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