Its about overall procedure,application and gating technique of flowcytometry in Stem cell enumeration

1. Hematopoietic Stem Cell Enumeration
By Flowcytometry
Susanta Das
Senior Cord Blood Bank Technician at Cordlife Sciences India Pvt.Ltd.

2. What is Flowcytometry
• “Flow Cytometry” as the name suggests is a technique for cell
counting and measurement of different properties of the cell with
the help of fluorescence.
• (‘Flow’=Fluid,‘cyto’=Cell, ‘metry’=count/Measurement)
• It is a laser based technology that measures and analyses different
physical and chemical properties of the cells/particles flowing in
a stream of fluid through a beam of light.

3. Components of Flow Cytometer
Flowcytometer
Fluidics:The fluidics system transports particles in a stream to the laser beam.
Optics: The optics system consists of lasers to illuminate the particles in the
sample stream and optical filters to direct the resulting light signals to the
appropriate detectors.
Electronics:The electronic system converts detected light signals into electronic signals
processed by computer.
Fluidics
Optics Electronics

4. Fluidics-Hydrodynamic Focusing
• When two streams of fluids with different flow rates are running side-by-side and in the same
direction into a flow cell, then a laminar flow is created. The central stream (sample stream) is
focused and surrounded by the secondary slower stream (sheath fluid). The sample pressure is
always greater than the sheath fluid pressure. It transport particles in a fluid stream to the laser
beam for interrogation.

5. Light Scatter-FSC & SSC
• Forward-scattered light (FSC) is proportional to cell-surface area or size.It is
detected in forward direction by photodiode.
• Side-scattered light (SSC) is proportional to cell granularity or internal complexity.
SSC is collected at approximately 90 degrees to the laser beam by a collection
lens.

6. Detection of cell with the help of size &
complexity
• Cell sub population based on FSC VS SSC.
• Major leucocyte sub population can be
differentiated using FSC and SSC
Particle size

7. Fluorescence
• A fluorescent compound absorbs light energy over a range of wavelengths that is
characteristic for that compound. This absorption of light causes an electron in the
fluorescent compound to be raised to a higher energy level. The excited electron
quickly decays to its ground state, emitting the excess energy as a photon of light.
This transition of energy is called fluorescence.

8. Fluoresce molecule and Cluster of
Differentiation
Cluster of differentiation(CD)-A cell surface marker which is present in
surface of the cell. It is different for different types of cells. It is used
for cell identification.
All leucocytes cell shows CD45+ and all hematopoietic progenitor cells
shows CD34+
With the help of this surface markers we use fluorescence molecule to
identify them
• FITC(Fluorescein iso thiocyanate)-it is tagged with antibody against
CD45+ cells.
• PE(phycoerythrin)-It is tagged with antibody against CD34+ cells.
• 7AAD(7 Amino actinomycin D)-It stained nucleus of dead cell. It helps
us to identify viable and non viable cell.

9. WORKING PROCEDURE
• Add 20µl of CD34/CD45 combo reagent and 7AAD viability dye in
Facs tube
• Add 100µl of Blood
• Incubate for 20mins & then add RBC lysing solution(pharmalyse)
• Incubate for 10 min
• Run the samples

10. Fluorescence spectra
• With the help of this three fluorochrome
we can distinguish CD45(FITC),CD34(PE),
• Viable and non viable cell(PerCp).
• CD45 = Leucocyte cells
• CD34+=Hematopoietic stem cells

11. Optical System
• The optical system consists of excitation optics and collection optics.
• Excitation Optics: The excitation optics consist of the laser and lenses that are used
to shape and focus the laser beam.

12. FLUROSCENCE DETECTORS(COLLECTION OPTICS)

13. Optical filters
• Optical filter: Once a cell or particle passes through the laser light, emitted SSC
and fluorescence signals are diverted to the photomultiplier tubes (PMTs) and a
photodiode collects the FSC signals.Optical filters placed infront of the PMTs for
specificity of detector.

14. Collection Lens
• Collection Lens: The collections optics consist of a collection lens to collect light emitted from the
particle–laser beam interaction and a system of optical mirrors and filters to route specified
wavelengths of the collected light to designated optical detectors.

15. Electronics-Signal detection
• Signal Detection: Light signals are generated as particles pass through the laser
beam in a fluid stream.These light signals are converted to electronic signals
(voltages) by photodetectors and then convert to the digital value by computer.

16. GATING STRATEGY OF FACS CANTO II

17. GATING STRATEGY OF FACS CALIBUR