This document summarizes the process of isolating peripheral blood mononuclear cells (PBMCs) from whole blood. It describes that PBMCs contain lymphocytes and monocytes that are important for the immune system. The process involves layering blood on top of ficoll-hypaque, then centrifuging to separate the buffy coat containing PBMCs from other blood components. The PBMCs are then cultured for research applications such as studying HIV infection of CD4+ cells.

1. ISOLATION OF PERIPHERAL BLOOD
MONONUCLEAR CELL (PBMC)

2. Blood is a specialized bodily fluid that delivers necessary
substances to the body's cells — such as nutrients and oxygen
— and transports waste products away from those same cells.
BLOOD
CONSTITUENTS OF HUMAN BLOOD
1. Cells
2. Plasma

3. CELLS
Red blood cells White blood cells
(RBCs or erythrocytes) (WBCs including leukocytes
and platelets also called
thrombocytes)
leukocytes thrombocytes
4.7 to 6.1 million (male), 4,000-11,000 200,000-500,000
4.2 to 5.4 million (female)

4. PLASMA
55% of whole blood is blood plasma --- fluid that is the
blood's liquid medium --- is straw-yellow in color
Plasma circulates dissolved nutrients, such as glucose,
amino acids, and fatty acids (dissolved in the blood or
bound to plasma proteins), and removes waste
products, such as, carbon dioxide, urea, and lactic acid

5. RED BLOOD CELLS
The most common type of blood cell and the body‘s
principal means of delivering oxygen to the body tissues
via the blood.
The cells are filled with hemoglobin, a biomolecule that can
bind to oxygen.
The blood's red color is due to the color of hemoglobin.

6. WHITE BLOOD CELLS
Cells of the immune system defending the body against
both infectious disease and foreign materials.
Five different and diverse types of leukocytes exist, but
they are all produced and derived from a multipotent cell
in the bone marrow known as a hematopoietic stem cell.
Leukocytes are found throughout the body, including the
blood and lymphatic system

7. WHITE BLOOD CELLS
White blood cells are often characterized as granulocytes or
agranulocytes
Granulocytes: characterised by the presence of differently
staining granules in their cytoplasm when viewed under
light microscopy. There are three types of granulocytes:
neutrophils, basophils, and eosinophils, which are named
according to their staining properties.
Agranulocytes: characterized by the apparent absence of
granules in their cytoplasm. The cells include
lymphocytes, monocytes, and macrophages.

8. Neutrophil 54-62%
Approx. % in
adults
Multilobed, faintly pink
Nucleus and granules
Eosinophil 1-6% Bilobed full of pink
Basophil 0% Bi or tri-lobed
Lymphocyte 25-33% Deeply stained
Monocyte 2-8% Kidney shaped

9. Peripheral Blood Mononuclear Cell
(PBMC)
Is a blood cell having a round nucleus, such as a
lymphocyte and monocyte.
These blood cells are a critical component in the immune
system to fight infection and adapt to intruders.
The lymphocyte population consists of T cells (CD4 and
CD8 positive ~75%), B cells and NK cells (~25%
combined).
PBMCs are widely used in research and clinical uses
every day. HIV research uses them because PBMCs
include CD4+ cells, the cells HIV infects.

10. B cells
B cells make antibodies
that bind to pathogens
to enable their
destruction.
T cells
CD4+ (helper) T cells CD8+ cytotoxic T cells are
co-ordinate the immune able to kill virus-infected
response and are important and tumor cells.
in the defense against
intracellular bacteria.
•Natural killer cells
•able to kill cells of the body
which are displaying a
signal to kill them, as they
have been infected by a
virus or have become
cancerous.

11. PBMCs are extracted from whole blood using ficoll, a
hydrophilic polysaccharide that separates layers of blood,
with monocytes and lymphocytes forming a buffy coat under a
layer of plasma after centrifugation.
This buffy coat contains the PBMCs.

12. Density Gradient Centrifugation
The basic idea behind the density gradient approach is
that the mixture of particles to be separated is placed
onto the surface of a vertical column of liquid, the
density of which progressively increases from top to
bottom, and then centrifuged.
The two main types of density gradient centrifugation
are rate-zonal separation and isopynic separation.

13. Density Gradient Centrifugation
Rate-Zonal Separation Isopycnic Separation
particles are separated particles migrate through
based on their size the solvent gradient until they
and mass reach the point where their
buoyant density is equal to
Proteins such as that of the gradient
antibodies and
virus particles

14. Blood collection
in EDTA vacutainer
PROCEDURE
Centrifuge at 1000rpm - 5 mins
Remove plasma add equal
volume of RPMI-1640
2ml of ficoll-hypaque
+ 3ml blood
Centrifuge 1000rpm - 30 mins Remove the buffy coat
add 2ml of RPMI-1640
Centrifuge 1000rpm 10 mins
Re - suspend the cells in
1ml RPMI-1640 and add to
tissue culture flask
containing 4ml RPMI-1640
Decant supernatent

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