Lab.

VI
Isolation and Separation of Leukocytes from
Human Peripheral Blood

Objectives
After completing this laboratory exercise you should be able to:

1. Isolate mononuclear cells from peripheral blood.

2. Purify adherent cells including B cells and monocytes.

3. Separate T cells by sheep RBC rosette formation.

Introduction

Human Blood

Mature immunocompetent cells can be found in secondary lymphoid organs. In

humans, peripheral blood is the most feasible source for such cells. A sample of human blood
is composed of ~50% plasma and the rest is cellular elements. The cellular elements include
erythrocytes (RBCs), leukocytes (WBCs) and platelets. Leukocytes constitute: 40-75%
neutrophils, 20-45% lymphocytes, 2-10% monocytes, 1-6% eosinophils and <1% basophils.
These cells are functionally and morphologically distinct, hence, they are categorized
according to their function and morphology. Morphological criteria include the uniformity of
the nucleus and the presence of granules in the cytoplasm. Leukocytes with multilobed-nuclei
are termed polymorphonuclear cells (PMNs) and cells with uniform single lobed-nuclei are
called mononuclear cells (MNCs). Also, leukocytes are classified according to granularity of
their cytoplasm into granulocytes which are cells with cytoplasmic granules and cells void of
cytoplasmic granules are called agranulocytes. Based on these categories, neutrophils,
eosinophils and basophils are considered as both granulocytes and PMNs while, monocytes
are granulocytes and MNCs, and lymphocytes are agranulocytes and MNCs. Moreover,
leukocytes have distinct functional differences, some are considered as members of the innate
immunity such as PMNs and monocytes and others contribute to the specific acquired
immunity. In addition, these cells are equipped with cell surface markers that allow them to do
their unique functions. Based on these differences in structure, morphology and function,
many techniques have been established to separate different leukocytes from each other.
In this exercise you will isolate lymphocytes from venous blood and learn how to separate and
enrich for T and B lymphocytes. The purified cells will be ready for enumeration and to be
used in functional assays.
In this Lab. you will isolate and separate leukocytes from human blood according to
differences in their densities, functional characteristics and cell surface markers.
Because blood is the source of many blood-born diseases (ex.: AIDS and hepatitis),
working with human samples creates a biological hazard. Thus, you should be wearing gloves
and be careful where you place the sample that you will take. Also, you should know how and
where you will dispose it when you are done.

Separation of PMNs from MNCs Using Density Gradient

Centrifugation
The principle behind density-gradient centrifugation relies on having two media with
different densities, one of them is a diluted blood sample and the other is a Ficoll solution.
The Ficoll solution has greater density than that of diluted human blood and when a
centrifugal force is applied, blood constituents will separate according to their densities.
Therefore, PMNs and RBCs will pellet at the bottom because they are denser than the rest of
blood components. Lymphocytes and monocytes will settle at the interface between blood and
Ficoll. Platelets and diluted plasma will remain in the upper layer since they are the least
dense.
Separation of Monocytes and B cells from other MNCs by

Adherence to Plastic or Glass Surfaces

The MNCs that you isolated from peripheral blood contain a mixture of B-cells, T cells,
monocytes and natural killer (NK) cells. Monocytes and B cells share the unique
characteristic of adhering to plastic and glass surfaces. This property is exploited for
separating these cells from non-adherent T cells and NK cells. Once separated from the mixed
population they can be further separated into B cells and monocytes depending on other
unique features. For example, the ability of monocytes to phagocytose nonspecifically can be
used to take iron particles, which allows them to be separated by magnetism from the rest of
non-phagocytic population of B cells. Another approach would be, the addition of magnetic
beads coated with antibodies against specific surface markers on the B cells such as the
human IgM B cell receptor or the CD16 on monocytes.
In this part of the exercise you will separate plastic-adherent cells in preparation for further
analysis.

Separation of T cells Using the Rosette Formation Method

The non-adherent cells that you isolated contain a mix of T cells as well as NK cells. The
following method utilizes the fact that T cells have a surface marker called CD2 that can
interact and bind with a specific marker on surface of sheep RBCs (sRBCs) consequently
called CD2 receptor (CD2R). Because sRBCs are feasible and readily visible, they provide a
neat tool for both enumeration and isolation of T cells from a mixed population. In this assay,
non-adherent cells are mixed with a sRBC solution allowed to bind to each other's and form a
flower-like aggregation having he T-cell at the center of the flower and the petals being
sRBCs. This configuration is termed rosette. Figure 2 depicts the rosette formation by T cells.
The rosettes can be used to count the number of T cells within a certain population. Also, they
can help in the isolation of these cells from a mixed population when the mixture is subjected
to density-gradient centrifugation.
Animals, Equipment, Materials and Reagent

Materials, equipment and reagents for bleeding of animals (see Lab. II).

Microzone electrophoresis cell, Beckman R101 or similar.

Power supply.

37ºC incubator or 100ºC oven.

Spectrophotometer or densitometers.

Cuvettes.

Sample applicator or micropipettes with tips.

Pipettes.

Parafilm and blotting paper.

Plastic foreceps. Plastic trays (7).

Glass plate (size slightly larger than cellulose acetate membrane).

Razor blades.

Sera of normal persons and patients. Sera of normal and immunized animals.

Barbital buffer, pH 8.6, 0.075M.

Cellulose acetate strips.

Ponceau S protein solution.

Washing solution (5% acetic acid).

Absolute ethanol.

Clearing solution (formic acid and dimethylsulfoxide, 1:9) or (30% cyclohexanone in

ethanol).
Exercise

1- Prepare serum samples you want to fractionate and label them sequentially (See
Exercise 3).
2- Set up the elecrtrophoresis apparatus. Follow the manufacturer's directions step by step
for proper use of the apparatus.
3- Fill the chambers with barbital buffer (pH 8.6) up to the desired level as set by the
manufacturer's instructions.
4- Use plastic forceps to remove the cellulose acetate strip and place it in the buffer in
one of the electrophoresis chambers and allow it to reach full saturation.
5- Take the membrane out and place it between two blotters to remove excess buffer.
6- Load membrane on bridge of the electrophoresis cell. Orient the reference hole on
strip at lower left of the bridge. Place the bridge on the cell making certain that the two
ends of the membrane are submerged within the buffer. Place the cell cover in
position.
7- Place a drop of each serum sample on parafilm. Cover with inverted beaker to avoid
evaporation of the serum. Load samples using a special sample applicator or a
micropipette (sample size 2ul). Rinse the sample applicator with distilled water after
the application of each sample, or use a new tip for each sample.
8- Connect the cell to power supply and adjust to 250 V and at 4-6mA. Run for 16-18
minutes then turn off the power supply and disconnect the cell.
9- Place the membrane in a tray containing Ponseau S dye/ fixative solution or Ponseau S
stain solution for 7 minutes. Meanwhile fill plastic trays with following solutions:
a. 100 ml rinse solution (5% acetic acid solution) -3 trays.
b. 100 ml dehydration solution (absolute ethanol) -1 tray.
c. 100 ml clearing solution (formic acid in diemethyl sufoxide or 30%
cyclohexanone in ethanol) - 1 tray.
10- Rinse membrane in 3 washes of 5% acetic acid solution until the dye no longer is
removed from the membrane.
11- Agitate the membrane in dehydration solution for one minute.
12- Place a clean glass plate in a tray containing clearing solution and transfer the
membrane on top of the glass plate. Agitate for 1 minute, then remove the glass plate
with the membrane on top of it.
13- Using a plastic forceps, remove excess clearing solution off the membrane.
14- Place glass plate and membrane in an oven preheated to 100c or allow the membrane
to dry in an oven at 37 c.
15- Using a razor blade, peel the membrane from the glass plate. Place the membrane in
clear plastic envelope and trim off the ends using sharp scissors.
16- Read protein fractions using a densitometer set at A520 and pre-warmed for 15
minutes.
17- Analyze the densitometer tracings of each fraction and calculate the relative
percentage of each.
18- If a densitometer is not available, the steps following step 10 above are as follows:
19- Place the membrane on clean glass plate and squeeze out excess fluid. Dry in an
incubator at 37ºC.
20- Use a razor blade to cut the strip into sections with each containing one distinct band
of protein. Place each section in a separate 13X 100-mm test tube.
21- Add 5 ml of clearing solution to each tube to dissolve the cellulose acetate membrane
of each section.
22- Standardize a spectrophometer set at A520 with clearing solution alone.
23- Determine the absorbance for each tube containing protein fractions and analyze
your results. Calculate the relative percentage of each fraction.