Cell separation is the process of removing one cell population from another within a biological sample, such as blood or tissue.

1. CELL SEPARATION
L E C T U R E R : S . P O R D E L
M . S C . O F I M M U N O L O G Y

2. What Is Cell Separation?

3. Cell separation:
is the process of removing one cell population from another within
a biological sample, such as blood or tissue.
Example uses of cell separation include:
•Capture of circulating tumor cells from blood
•Isolation of immune cells from peripheral blood
•…

4. Immune cell isolation refers to methods that enable the enrichment of immune cell
subsets by techniques such as:
 erythrocyte lysis
 magnetic isolation
 density centrifugation
 Sorting by flowcytometry
Cell Separation:

5.  This MACS technology is based on MACS Microbeads (Antibodies coupled with Magnectic
Beads), MACS Separator and the MACS Columns.
 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.
Magnetic-activated cell sorting (MACS)

7.  Target cells that are
labelled with MACS
Microbeads are attracted to
the column walls by the
magnetic force (Positive
Selection) and Unlabelled
cells (Negative Selection).

8. 1 2 3
Isolation of mononuclear cells from human peripheral blood by Ficoll-Paque density gradient.
density centrifugation

10. F L O W C Y T O M E T R Y
L E C T U R E R : S . P O R D E L
M . S C . O F I M M U N O L O G Y

11. •Flow ~ cells in motion
•Cyto ~ cell
•Metry ~ measure
Measuring properties of cells while in
a fluid stream
WHAT IS FLOWCYTOMETRY?

12. • Clinical
•Research

13. How it works
1. Draw cells, with excess fluid, from test tube into machine.
2. Cells pass in single file past laser.
3. Laser hits cell and light is scattered.
4. Photomultiplier multiplies light intensity and a light sensor
measures the amount of light and scatter pattern.
5. Phenotyping, Size and granularity detection

14. Basics of Flowcytometry:
Fluidics: cells in suspension flow in single-file
through
Optics: an illuminated volume where cells scatter light and
emit fluorescence that is collected, filtered
and
Electronics: fluorescence converted to digital values that are
stored on a computer

15. How Is It Done:
Hydrodynamic Focusing

16. Size and granularity detection

17. WHY LOOK AT FSC V. SSC
• Since FSC ~ size and SSC ~ internal structure, a correlated
measurement between them can allow for differentiation of
cell types in a heterogenous cell population
FSC
SSC
Lymphocytes
Monocytes
Granulocytes
RBCs, Debris,
Dead Cells

18. +
Incubate Acquire
Phenotyping

19. Dual parameter:Single parameters:

20. Principles Of Fluorescence

21. Laser
Light
488nm
FL2 PE
SSC
FSC
Fluidics
Electronics
Computer
FL3 PerCP, Cy5
FL1 FITC
A cytometer consists of:
Light detectors

22. Dot plot chart

23. Basics of Flowcytometry:
Fluidics: cells in suspension flow in single-file
through
Optics: an illuminated volume where cells scatter light and
emit fluorescence that is collected, filtered
and
Electronics: fluorescence converted to digital values that are
stored on a computer

24. Flow Cytometry Is A Powerful Technique For Characterizing Immune
Cells
• Allows For Detection Of Surface Markers Of Cells
• Allows For Detection Of Intracellular Factors
• Allows Detection Of Secreted Factors By Cells
• Allows For Detection Of DNA Content

25. Immune cell isolation refers to methods that enable the enrichment of immune cell
subsets by techniques such as:
 erythrocyte lysis
 magnetic isolation
 density centrifugation
 Sorting by flowcytometry
Cell Separation: