Flow Cytometry

It measures multiple physical characteristics (size, granularity, fluorescence) of single cells as they flow in suspension through a laser-interrogating system.

What is the core function of a flow cytometer?

sorting flow cytometry

isolates desired cells for further use (e.g. CD4+ T-cells for DNA extraction)

non-sorting flow cytometry

analyzes cell properties without physically separating them

Cytoflex LX (analyzer) and Cytoflex SRT (cell sorter)

What machine brands are hosted at UP Manila under PhilDIAMOND?

tissues (e.g., liver, tumor), microorganisms, and nuclei

Besides PBMCs, what other samples can be analyzed using flow cytometry?

Gentle maceration or enzymatic digestion (e.g., trypsinization) to break cell adhesions.

What techniques can make tissues analyzable by flow cytometry?

forward scatter (FSC); larger cells produce greater forward scatter.

How is cell size measured in flow cytometry?

side scatter (SSC); more granular cells scatter light at 90° more strongly

How is granularity measured?

Autofluorescence and staining with fluorochrome-conjugated antibodies.

What causes cells to fluoresce during flow cytometry?

To differentiate cell types based on side scatter (granularity) vs forward scatter (size).

What is a “dot plot” in flow cytometry used for?

X-axis

represents Fluorescence intensity

Y-axis

represents Number of events detected with that intensity.

fluorochromes

When excited by a laser within their absorption spectrum, they emit fluorescence detected by filters specific to their emission spectrum.

To avoid signal confusion—each marker must be tagged with a unique fluorochrome to be individually detected.

Why are different fluorochromes needed for different markers?

Fluorescent proteins (e.g., GFP)
Synthetic small molecules (e.g., FITC)
Quantum dots (e.g., Qdots)
Polymer dyes (e.g., Brilliant Violets)
Tandem dyes (e.g., PE-Cy5)

List the 5 classes of fluorochromes

Use bright fluorochromes for rare markers and dim fluorochromes for abundant ones.

When should bright vs dim fluorochromes be used?

spectral overlap

It occurs when the emission spectra of two fluorochromes overlap, making it hard to distinguish them.

color compensation

By subtracting a fraction of one fluorochrome's signal from the other using mathematical algorithms in the software.

Use fewer or more distinct fluorochromes, avoid unnecessary markers, and design experiments efficiently.

How can spectral overlap be minimized?

By detecting markers like immune checkpoint inhibitors (PD-1, TIM3, TIGIT, LAG3) to evaluate T-cell function.

How is flow cytometry used in T-cell activation/exhaustion analysis?

CD8+: Hierarchical loss of function; CD4+: Reduced cytokine production and support.

What are the key differences in CD4+ and CD8+ T cell exhaustion?

Naive
Central Memory
Effector Memory
Transitional Memory
Terminally Differentiated

What are the 5 T-cell subsets identified using 3 markers?

By identifying correlation between immune checkpoint expression and cognitive test performance.

How can flow cytometry relate to neurocognitive impairment in HIV+ patients?

Marker identification, target validation, and high-throughput screening of drug candidates.

What are the applications of flow cytometry in early drug discovery?

high-throughput drug screening

Automated analysis of large sample volumes using robotics and sensitive detection tools to evaluate drug effects

For disease modeling, proof-of-concept testing, and in vitro/in vivo assays.

How is flow cytometry applied in preclinical studies?

A negative fluorescence result (no detection of the target).

What does a histogram peak on the left side and none on the right indicate?

The machine reads fluorochromes, not markers. Same color = indistinguishable signal.

Why can't two markers use the same fluorochrome?