Immunology Course 2023

16 Dec 2023

Workshop · Flow Cytometry

December 16, 2023

Courtesy: Vinod Kumar Dorai (PhD student, Immunology Lab, IISc)

When studying specific cells in flow or being transported:

Develop a device to sample blood.
Understanding individual healing processes in different individuals.

One approach to study healing variations:

Biopsy is not preferred.
Create a system involving blood sampling, antibody binding to specific proteins, utilizing various antibody clones for the same protein.

Flow Cytometry

Microfluidics Design: Utilizes a fluid system to narrow down cells, allowing only one cell to pass through.
Each cell passes through a laser for analysis.
Antibody-tagged cells get excited, emitting a signal for identification.
Tracking cell kinetics involves injecting mice with a synthetic nucleotide (Edu) marking newly generated cells.
Differentiates between preexisting and newly generated neutrophils.

Basics:

Technology Overview: Rapid analysis of cells or particles flowing past lasers.
Cells analyzed for visible light scatter and fluorescence parameters.
Multiple lasers used for various fluorophores.

How Does it Work:

Measures visible light scatter in forward and side directions.
FSC (Forward Scatter) indicates cell size.
SSC (Side Scatter) detects granular properties (e.g., granulocytes).
Samples prepared for fluorescent measurement via transfection and expression of fluorescent proteins (e.g., GFP).
Differentiating live and dead cells using propidium iodide.

Importance of Quantifying Dead and Live Cells:

Dead cells exhibit distinct surface marker properties compared to live cells.

Fluidics

Controlled fluid dynamics to allow single-cell analysis.
Concepts of fluid dynamics applied to ensure single-cell passage.
Varying flow rates for sensitivity adjustments and high-throughput analysis.
High throughput aids in immunotyping without compromising sensitivity.

Challenges:

Managing heat generation and the number of events analyzed.

Filters

Long Pass Filters:
Band Pass Filters:

Detectors

Photodiodes: Utilized in forward scattering, requiring sufficient photons.
PMT (Photomultiplier Tube): Amplifies energy received from photons, providing enhanced signal detection and calibration capabilities.

Compensation Theory

Addressing spillover issues arising from multiple fluorochromes used.
Overlapping wavelength ranges make it challenging to isolate light from specific fluorochromes.

Multicolor Panel Designing

Factors to consider:
- Selection of surface and intracellular markers.
- Optimal antibody concentrations.
Steps involved in panel designing:
- Understand markers and study objectives.
- Decide marker combinations and concentrations for different cell types.

Enhancing understanding through panel design:

Identification markers (4) and activation markers (4) required.
Combination of these markers is crucial for effective panel design.