Peripheral

Blood Mononuclear Cells (PBMCs)

Principle
Human blood consists of equal parts of blood plasma and
blood cells. These include erythrocytes (red blood cells),
leukocytes (white blood cells) and thrombocytes (platelets).
Leukocytes are further subdivided into different cell types. These
include lymphocytes and monocytes, which (in cooperation with
other cells) form the basis of the innate immune system and
which, owing to their single nucleus, are referred to as peripheral
blood mononuclear cells (PBMC). The term lymphocyte
encompasses two major classes, B-lymphocytes and T-
lymphocytes. B-lymphocytes are responsible for antibody
production, whereas T-lymphocytes produce signal molecules
which will ultimately lead to the removal of diseased or foreign
cells.
Lymphocytes are isolated from “buffy coats” (whole
blood concentrates without serum). PBMCs can be separated from
other components of the blood, such as erythrocytes and
granulocytes, via density gradient centrifugation using Ficoll-
Paque PLUS. Ficoll-Paque has a density of 1.007 g/mL. Due to
their higher density, erythrocytes, granulocytes and dead cells will
pass through the Ficoll layer, whereas lymphocytes and
monocytes, based on their lower density, will accumulate at the
plasma-gradient boundary

Materials
 Anti-coagulated blood
 Ficoll-hypaque
 Hank’s buffer
 Centrifuge tubes
 Glass beaker
 Glass Pasteur pipets
 Centrifuge
 Microscope

Procedure
1. Carefully layer 2 mL of diluted cell suspension over 2mL of
Ficoll in a 5 mL tube.
2. Centrifuge at 2000RPM 20 minutes at 20 °C in a
swinging- bucket rotor without brake.
3. Aspirate the upper layer leaving the mononuclear cell
layer (lymphocytes, monocytes…) undisturbed at the
interphase.
4. Carefully transfer the mononuclear cell layer to a new
tube.
5. Fill the tube with buffer, mix, and centrifuge at 1500RPM
for 10 minutes at 20 °C. Carefully remove supernatant
completely.
6. Resuspend the cells with buffer and then observe it with
microscopy.
Result

Analysis
PBMC’s are separated and can be used for other
experimental procedures.

Phagocytosis test
Principle
Phagocytes ingest and destroy foreign particles (such as
bacteria) and dead host cells. Phagocytes form a vitally
important front-line defense against infection and are a
major part of the innate immunity. Phagocytic cells include
neutrophils and macrophages. The major phagocytic cell of
the blood stream is the neutrophil. The major phagocytic
cell of the tissues is the macrophage. Macrophages arise
from monocytes in the bloodstream. Phagocytes migrate out
of the blood vessels to infection sites where neutrophils and
macrophages phagocytes pathogens and clear out the
infection.

In the first step of phagocytosis, phagocytes are

attracted by and move toward a variety of substances
generated in immune response; this process is called
chemotaxis. The next step in phagocytosis is the adherence
of the antigen to the cell membrane of the phagocytic cells.
Adherence induces membrane protrusions, called
pseudopodia, to extend around the attached material and to
ingest them. Phagocytic cells contain various receptors which
help them to attach with bacteria/virus. Following
attachment, polymerization and then depolymerization of
actin filaments send pseudopods out to engulf the microbe.
 Then the phagosome moves toward the cell interior, where it
fuses with a lysosome to form a phagolysosome. Lysosomes
contain lysozyme and variety antimicrobial and cytotoxic
substances that can destroy phagocytosed microorganisms
and cells, finally The digested contents of the phagolysosome
are then eliminated in a process called exocytosis.

Materials
 Staphylococcus suspension(70 degree Celsius 0.5h)
 Acid citrate dextrose treated blood
 Wright stain, double distilled water
 Glass tubes, glass Pasteur pipets, slides
 Incubator, staining rack, microscope

Procedures
1. Add 0.5ml anticoagulant blood into a glass tube, add
another 0.25ml pretreated staphylococcus and mix
2. Place the glass tube into incubator,37 degree Celsius
for 30min
3. Take out the glass tube, and mix carefully with Pasteur
pipet
4. Make the blood smear
5. [lace thoroughly dried blood film on an appropriate
staining rack
6. Flood slide with several drops of wright stain
7. After 1 minutes, add an equal volume of ddw, and mix
thoroughly by gently blowing on slide
8. After 5minutes, thoroughly rinse with running water
and air dry
9. Search the neutrophils with phagocytized
staphylococcus under the microscope
Result
 Phagocytosis by neutrophil when blood films are
stained using wright stain, nuclei will be purple,
cytoplasm, blue to light pink, and red blood cells,
pink to orange
 Phagocytosis by macrophage will be macrophages
with phagocytized chicken red blood cells

Name: Kassa Megzemwork Kalayu

Student id: 185019011