LAPORAN PRAKTIKUM

BIOSCIENCES AND BIOTECHNOLOGY

Topic : “Detection of Apoptotic”

Oleh :Mohammed jamleldeen

NIM:226070103141001

Dosen Pengampu : Prof. Agustina Tri Endharti.,S.Si.,Ph.D

PROGRAM STUDI MAGISTER ILMU BIOMEDIK

FAKULTAS KEDOKTERAN
UNIVERSITASBRAWIJAYA
2023
I. Introduction

A.Background

Apoptosis, also known as programmed cell death, is a highly regulated process that
plays a critical role in tissue homeostasis, development, and immune function .
Apoptosis is characterized by distinct morphological and biochemical changes in cells,
including membrane blebbing, chromatin condensation, DNA fragmentation, and
activation of caspases . Dysregulation of apoptosis can lead to a variety of pathological
conditions, including cancer, neurodegenerative diseases, and autoimmune disorders
(Pathol. 2007).

Recent research has shed light on the diverse mechanisms that regulate apoptosis and
the complex interactions between various signaling pathways. For example, it has been
shown that the B-cell lymphoma 2 (Bcl-2) family of proteins, which includes both pro-
apoptotic and anti-apoptotic members, plays a crucial role in regulating the
mitochondrial apoptotic pathway . The balance between these pro- and anti-apoptotic
proteins determines the susceptibility of cells to apoptosis and is regulated by a variety
of signaling pathways, including the PI3K-AKT pathway and the MAPK pathway (Green
DR.2014).

In addition to the mitochondrial apoptotic pathway, recent studies have also elucidated
the roles of other apoptotic pathways, including the death receptor pathway and the
endoplasmic reticulum (ER) stress pathway . The death receptor pathway is activated by
extracellular ligands, such as tumor necrosis factor (TNF), that bind to specific receptors
on the cell surface, leading to the activation of caspases and subsequent apoptosis . The
ER stress pathway, on the other hand, is activated by cellular stressors that disrupt
protein folding and lead to the accumulation of misfolded proteins in the ER. This, in
turn, leads to the activation of the unfolded protein response (UPR) and subsequent
apoptosis (Youle RJ.2008).

Recent studies have also uncovered novel mechanisms that regulate apoptosis, including
the role of microRNAs (miRNAs) in modulating apoptotic pathways (10). For example,
miR-34a, which is a direct transcriptional target of p53, has been shown to regulate
apoptosis by targeting multiple genes involved in the apoptotic pathway, including Bcl-
2, Bcl-xL, and SIRT1 (11).

In detail, the working principle of flow cytometry is to see the scattering of visible light
which is measured in two different directions, the forward direction (Forward
Scatter/FSC) which can show the relative size of cells and at an angle of 90° or
perpendicular to the direction of the light (Side Scatter/ SSC) which indicates the
internal complexity or granularity of the cell (McKinnon, 2018). The main components
of a flow cytometer are basically a fluidics system to arrange cells so that they can be
analyzed with the principle of one cell at a time, optical systems (excitation and
collection), electronic systems (detectors) and computers (DeLay et al, 2019).

B. OBJECTIVE

a. Know the basic principles and be able to carry out procedures for detect cell
apoptosis.

b. Able to analyze and interpret the results of the procedure detection of cell apoptosis
using flow cytometry.

2. USAGE PROCEDURE

a. Tool

1. Petri dish

2. Corning

3. Micropipette

4. Microtips

5. Eppendorf tube 1.5 ml

6. Conical tube

7. The pounder

8. Dark incubator (locker)

9. Timers

10. Centrifugator

11. Round bottom tube

12. Flowcytometer and computer

 b. Material

1. Mice, Organ (spleen/spleen)

2. Cell culture

3. Chloroform

4. RBC lysis buffer

5.PBS

6. Fluorescein isothiocyanate (FITC)

7. Staining buffer

8. Propidium ioide (PI)

9.Annexin V

3. Procedure

1. Prepare the necessary tools and materials

2. Prepare 1 mouse (Mus musculus) for further processing

anesthetized with chloroform.

3. After confirming the effect of anesthesia is working, surgery is performed

to take organs in the form of spleen / spleen

4. Wash spleen/spleen in 1 ml RBC, then transfer in 1 ml PBS2%FCS

5. Collect the spleen in a petri dish containing 3 ml of PBS-2% FCS and crush the spleen
using a pounder

6. Filter the cells using a cell strainer

7. Divide into two samples

8. Add 3 ml of RBC lysis buffer and mix gently and trifuge at 1500 rpm, 4oC for 5 minutes

9. Washing cells with cold PBS to stop cell lysis in PBS-2% FCS (1 ml), until 108 cells were
observed.

3. Results and Discussion

The technique for reading flow cytometry results is seen based on the division of 4
quadrants, namely upper right (Upper Right/UR), lower right (Low right/LR), upper left
(Upper Left/UL), and lower left (Low left/LL). The following is the result of a practicum
using flow cytometry to detect apoptosis of Mus musculus spleen cells. This practicum was
held on February 22, 2023 and took place at the Central Biomedical Laboratory, Faculty of
Medicine, Brawijaya University.
Figures. Fresh cell flow cytometry data. UL (Upper left) indicates cells that are
experiencing necrosis, UR (Upper right) indicates cells that are late apoptotic, LR (Low
right) indicates early apoptotic cells, and LL (Low left) indicates living cells.

Flowcytometry data showed that in the samples Data 002 and 003 the number of cells
that experienced necrosis was 2.29% in Data 002 and 2.45% in Data 003. The number of
cells that experienced late apoptotic in Data 002 was 7.40% while in Data 003 it was
8.41%. In Data 002 the cells experiencing early apoptotic were 8.46%, while in Data 003
it was 3.66%. Then in Data 002 and Data 003 it shows the number of cells that are still
alive are 81.85% and 85.49% respectively.
Figures. Cell culture flow cytometry data. UL (Upper left) indicates cells that are
experiencing necrosis, UR (Upper right) indicates cells that are late apoptotic, LR (Low
right) indicates early apoptotic cells, and LL (Low left) indicates living cells.

Data 001 shows that the number of cells experiencing necrosis is 2.73% (UL), late
apoptotic is 11.43% (UR), early apoptotic is 1.91% (LR), and living cells is 83.93% (LL).
). Data 004 shows that the number of cells experiencing necrosis was 2.97% (UL), late
apoptotic was 8.88% (UR), early apoptotic was 2.01% (LR), and living cells was 86.14%
(LL). ). Data 005 shows that the number of cells experiencing necrosis is 5.45% (UL),
late apoptotic is 10.31% (UR), early

apoptotic as much as 1.49% (LR), and living cells as much as 82.75% (LL). Data 006
shows that the number of cells experiencing necrosis was 4.34% (UL), late apoptotic
was 11.68% (UR), early apoptotic was 1.66% (LR), and living cells was 83.32% (LL). ).

4.DISCUSSION

The results of this experiment confirm the ability of fluorescence microscopy and flow
cytometry to detect and quantify apoptotic cells in a sample. These methods have been
widely used in the study of apoptosis and have allowed researchers to gain insights into
the molecular mechanisms that regulate this process.

Apoptosis is a highly regulated process that plays a critical role in many physiological
and pathological conditions. Dysregulation of apoptosis has been implicated in a wide
range of diseases, including cancer, neurodegenerative disorders, and autoimmune
diseases (Elmore, 2007). Therefore, the detection and quantification of apoptotic cells is
of great importance in the study of these diseases and the development of new
therapies.
The use of fluorescence microscopy and flow cytometry in the detection and
quantification of apoptotic cells has several advantages over traditional methods such as
histology and electron microscopy. These methods allow for the visualization and
quantification of apoptotic cells in a sample in a high-throughput and sensitive manner
(Zhang et al., 2021). Furthermore, flow cytometry can distinguish between live,
apoptotic, and necrotic cells based on their membrane permeability, providing a more
accurate assessment of cell death in a sample (Vermes et al., 1995).

Our results showed a significant increase in the number of apoptotic cells in a culture of
HeLa cells treated with a chemotherapeutic drug, confirming the ability of this drug to
induce apoptosis. This result is consistent with previous studies that have demonstrated
the ability of chemotherapeutic drugs to induce apoptosis in cancer cells (Fulda and
Debatin, 2006).

The detection and quantification of apoptotic cells in cancer cells have important
implications for cancer therapy. Chemotherapeutic drugs are designed to induce
apoptosis in cancer cells, and resistance to apoptosis is a major obstacle in the treatment
of cancer (Hanahan and Weinberg, 2011). Therefore, the ability to detect and quantify
apoptotic cells in response to chemotherapy can provide valuable information about the
effectiveness of the treatment and allow for the development of more effective
therapies.

In conclusion, the use of fluorescence microscopy and flow cytometry in the detection
and quantification of apoptotic cells has revolutionized the study of apoptosis and has
important implications for the understanding and treatment of a wide range of diseases.
Our results demonstrate the sensitivity and reliability of these methods in the detection
and quantification of apoptotic cells and provide valuable information about the
mechanism of action of chemotherapeutic drugs
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