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Hematology

PBMC Isolation using The Miltenyi Biotec MACS Cell Separation Technology

PBMC Isolation using The Miltenyi Biotec MACS Cell Separation Technology

The Miltenyi Biotec MACS Cell Separation Technology is
one of the most prominent powerful tool for specific cell separation. This MACS
technology is based on MACS Microbeads (Antibodies coupled with
Magnectic Beads), MACS
Separator and the MACS Columns.
MACS MicroBeads are superparamagnetic antibody
particles that are approximately 50
nanometers in diameter and are composed of a biodegradable matrix.
Furthermore it is manufactured in a manner that does not detrimentally affect
nor modify the alter structure, function, or activity status of
labeled cells. Hence,
it does not interfere with the subsequent downstream experimental processes and
is not necessary to remove them from cells after the separation process. 

The MACS
separation process occurs within the MACS Columns. The MACS Separator acts as a
“magnetic bar” by inducing a strong permanent high-gradient magnetic field into
the column matrix. Target cells that are labelled with MACS
Microbeads are attracted
to the column walls by the magnetic force (Positive Selection). Unlabelled
cells (Negative Selection), are allowed to flow and pass through to be
collected at the other end via natural gravitational force. Hence, with MACS
technology, both Target and non-Target cell fractions can be collected and
isolated with high purity to be subjected to downstream experimental procedures
immediately. 

* A video link is provided for a more clearer picture of the manual method of this Miltenyi Biotec MACS Cell Separation Technology: http://www.youtube.com/watch?v=6vyj8-OkOKc.
Watch it with your own discretion as research and experimental aims may differ. 

Process of Miltenyi Biotec MACS Cell Separation Technology

Magnetic Labeling 

Highly purified PBMCs can be isolated from whole blood via the procedure in the previous chapter.
Subsequently the PBMCs are re-suspended in the appropriate amounts of
AutoMACS Buffer
depending on individual protocol and incubated with the desired MACS Microbeads
that is specific to the antigen on the Target cells. The incubation process is
usually
atleast 15minutes to
a maximum of 30 minutes in the optimal temperature of 4
°C usually in a refrigerator.

Next, the sample is then resuspended further with the
appropriate amount of AutoMACS Buffer and subjected to washing. This washing
step is done by first centrifuging the sample and discard the supernatant to
remove any unbound MACS
microbeads, leaving a PBMC cell pellet. This PBMC cell pellet is then subjected to
the subsequent MACS separation procedure.

Magnetic Separation
(Part 1)

After the PBMC cell pellet is resuspended with the
appropriate volume of AutoMACS buffer, the cell suspension is then
pipetted into the AutoMACS
Column to allow it to flow through in a drop-wise manner via natural
gravitational force. Target cells that are labelled with the antibody coupled
with magnetic beads will get attracted onto the side of the AutoMACS Column via
magnetic attraction forces produced by the magnetic separator. On the other
hand, non-target cells that are not labelled, will flow through into a
collection tube as the negative fraction.

Magnetic Separation
(Part 2)

After the first
initial cell suspension have fully flowed through, the AutoMACS Column is then
subjected to washing to make sure that all non-target cells are washed into the
collection tube, leaving a purified positive fraction in the walls of the
column. The washing step is a procedure performed simply by adding AutoMACS
buffer into the AutoMACS column and leave it to flow through. The number of
washing steps and the volume of AutoMACS Buffer varies with different protocol.

Elution of the
labelled target cell fraction

After the
completion of the washing step, a plunger is then used to “push” all the target
cells that are attracted on to the walls of the
AutoMACs Column. This is done by first removing the AutoMACS column
from the Magnetic Separator, proceeding with
pipetting the desired volume of AutoMACS Buffer into the top of the
column and subsequently use the plunger provided to “push” all the remaining
contents into a collection tube. The collection tube will now contain the
positively selected target cell fraction.

Purification of the eluted positively selected target cell fraction

The eluted positively selected target cell fraction can now be further
purified by subjected to a washing step to remove any impurities in the cell
suspension. This is done by centrifuging the cell suspension and remove the
supernatant leaving the highly purified target cell pellet at the bottom of the
collection tube, allowing further downstream experimental procedures.

Similarly, the
negatively selected fraction can also be purified using this method to obtain
the cell pellet for downstream research procedures

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