week 10 - Imaging T cell function Flashcards by Anya James

Imaging T cell function
T cell modulation in Tumour micro environment

T cell infiltration into soild tumours face challenges
o Physical barriers and TME
o T cell dysfunction and exhaustion
o Tumour antigen heterogeneity

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IMAGING T CELL FUNCTION
Cell labelling strategies

Cell labelling strategies

Emit energy as photons
Stains to label specific part of cell

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Q: What are Vybrant dyes?

Long-chain dialkylcarbocyanine dyes
Used to label cells by inserting into lipid membranes

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Why Do Vybrant Dyes Insert into Membranes?

Q: Why can Vybrant dyes label cell membranes?

They are amphipathic
→ Interact with both hydrophobic lipid tails and hydrophilic environments

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Nature of Binding

Q: How do Vybrant dyes associate with membranes?

Non-covalent insertion into lipid bilayer
Can laterally diffuse within membranes

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Inheritance Through Cell Division

Q: Why are Vybrant dyes useful in T cell proliferation studies?

Dye is passed to daughter cells
Allows tracking of cell division over generations

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

In Vivo Applications

Q: Where are Vybrant dyes used in vivo?

Cell tracking in:

Mice
Zebrafish

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Staining Properties

Q: What are the staining characteristics of Vybrant dyes?

Strong, uniform membrane staining
Effective at low concentrations
Generally low cytotoxicity (cell-dependent)

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Limitations

Q: What is a limitation of Vybrant dye use?

Cytotoxicity and performance can vary by cell line

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Imaging Duration

Q: How long can Vybrant dyes be used for imaging?

Enable long-term imaging (~2 days) after labelling

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Types of Vybrant Dyes

Q: What are common Vybrant dyes?

DiO
DiI
DiD

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Fluorescence Properties (Diagram-Based)

Q: How do DiO, DiI, and DiD differ?

Different fluorescence emission wavelengths:

DiO: shorter wavelength (green)
DiI: intermediate (orange/red)
DiD: longer wavelength (far-red)

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IMAGING T CELL FUNCTION
Cell labelling strategies
Vybrant Dyes

Key Advantage

Q: What is the main advantage of Vybrant dyes in cell biology?

Allow non-invasive, long-term tracking of living cells

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Q: What is Hoechst 33342?

A bis-benzimidazole fluorescent dye
Binds to double-stranded DNA (dsDNA)
Used for nuclear staining and cell tracking

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

DNA Binding Mechanism

Q: How does Hoechst 33342 bind DNA?

Binds to the minor groove of DNA
Causes a conformational change
Leads to fluorescence emission

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Key Property

Q: Why is Hoechst 33342 useful for imaging?

High affinity for DNA
Produces a bright nuclear signal

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Cell Permeability

Q: Why can Hoechst 33342 enter live cells?

More lipophilic (N-cap structure)
Can cross the cell membrane

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Hoechst vs DAPI

Q: How does Hoechst 33342 differ from DAPI?

Hoechst: cell-permeable → stains live cells
DAPI: not membrane permeable → stains fixed cells only

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Imaging Use

Q: When is Hoechst 33342 best used?

Fixed cells
Short-term live-cell imaging

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Limitations – DNA Damage

Q: What are the drawbacks of Hoechst 33342?

Interferes with:
DNA replication
Transcription
Can cause DNA damage
Inhibits cell proliferation

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Signal Stability

Q: Why is Hoechst 33342 not ideal for long-term imaging?

Signal decreases over time
Not suitable for tracking over many cell divisions

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Key Trade-Off

Q: What is the main trade-off when using Hoechst 33342?

Strong nuclear staining
❌ But potential toxicity and limited long-term use

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IMAGING T CELL FUNCTION
Cell labelling strategies
Hoechst 33342

Big Picture

Q: When would you choose Hoechst 33342 over membrane dyes?

When you need clear nuclear staining
For short-term imaging or fixed samples
Not for long-term proliferation studies

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IMAGING T CELL FUNCTION
Cell labelling strategies
BODIPY Dyes

Q: What are BODIPY dyes?

A class of fluorophores used for cellular imaging
Known for bright and stable fluorescence

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IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Q: What are the main properties of BODIPY dyes?

Intense fluorescence High photostability Tuneable spectral properties (can adjust emission wavelengths)

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Functional Uses of BODIPY Q: How are BODIPY dyes used in biological imaging?

Can be conjugated to molecules to target specific structures Used as functional probes

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes BODIPY as a pH Probe Q: How does BODIPY function as a pH probe?

Fluorescence changes depending on pH Used to monitor cellular environments

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Lipid Imaging Application Q: How is BODIPY used to study lipid accumulation?

Conjugated to cholesteryl esters Visualises lipid accumulation (e.g. in zebrafish models)

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Golgi Apparatus Staining Q: How can BODIPY be used to stain organelles?

Conjugated to ceramide Specifically stains the Golgi apparatus

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Fluorescence Sensitivity Q: What happens to BODIPY fluorescence when pH changes?

Fluorescence properties change → Enables detection of cellular conditions

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Limitations in Imaging Q: What is a limitation of BODIPY dyes for live-cell imaging?

Non-covalent imaging is not stable long-term Typically limited to a few hours

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Big Picture Q: When would you choose BODIPY dyes over other probes?

When you need: High-quality fluorescence Organelle-specific targeting Functional readouts (e.g. pH, lipids)

IMAGING T CELL FUNCTION Cell labelling strategies BODIPY Dyes Key Advantage Q: What makes BODIPY dyes particularly useful?

Combine brightness + stability + flexibility Ideal for targeted, short-term functional imaging

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Q: What is covalent labelling in cell imaging?

Permanent attachment of probes to cellular molecules (e.g. proteins) Enables intracellular retention of the label

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Key Advantage Q: Why is covalent labelling useful?

Long retention time Signal is passed to daughter cells during division

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Intracellular Retention Mechanism Q: How are dyes retained inside cells in covalent labelling?

Form covalent bonds with intracellular proteins or glycans Prevents dye leakage

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Maleimide Labelling Q: How does maleimide labelling work?

Reacts with thiol (-SH) groups of cysteine residues Optimal at pH 6.5–7.5

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Target of Maleimide Reaction Q: Which amino acid is targeted by maleimide dyes?

Cysteine

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling NHS Ester Labelling Q: How does NHS (succinimidyl ester) labelling work?

Reacts with primary amines (-NH₂) Found on lysine residues

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Target of NHS Reaction Q: Which amino acid is targeted by NHS esters?

Lysine

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling NHS Reaction Conditions Q: At what pH does NHS ester labelling occur?

pH 7–9

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Reaction Mechanism Insight Q: Why are lysine residues highly reactive in NHS labelling?

The amine group attacks an electrophilic carbon Leads to stable covalent bond formation

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Timing of Labelling Q: When can covalent labelling be applied in experiments?

Before or after infection Flexible depending on experimental design

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Proliferation Dyes Q: What dyes are used to track cell proliferation?

CFSE CFDA-SE

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling CFSE Mechanism Q: How does CFSE become fluorescent inside cells?

Initially non-fluorescent Intracellular esterases cleave diacetate groups Produces fluorescent, protein-binding dye

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling CFSE and Cell Division Q: How does CFSE track cell proliferation?

Dye is equally divided between daughter cells Fluorescence intensity halves with each division

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Glycan-Based Covalent Labelling Q: How can covalent labelling occur via glycans?

Through metabolic glycan engineering Targets cell surface glycoproteins

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Step 1 – Metabolic Glycoengineering Q: What happens in the first step of glycan labelling?

Cells take up Ac₄ManNAz (azide-modified sugar) Incorporated into cell surface glycans

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Step 2 – Click Chemistry Q: How are probes attached in glycan labelling?

DBCO probes react with azide groups Via SPAAC (click chemistry) Forms covalent bond

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Why Use Glycan Labelling? Q: What is the advantage of glycan-based covalent labelling?

Stable cell surface labelling Long-term tracking of cells

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Big Picture Q: What are the main strategies of covalent labelling?

1. Protein targeting (cysteine, lysine) 2. Proliferation dyes (CFSE) 3. Glycan labelling (click chemistry)

IMAGING T CELL FUNCTION Cell labelling strategies Covalent Labelling Key Comparison Q: How does covalent labelling compare to non-covalent labelling?

Covalent: long-lasting, stable, inherited Non-covalent: reversible, shorter-term

IMAGING T CELL FUNCTION Cell labelling strategies Why Metabolic Labelling? Q: Why is metabolic labelling used in cell tracking?

Avoids cytotoxicity of other labelling methods Enables stable, long-term cell tracking

IMAGING T CELL FUNCTION Cell labelling strategies What is Metabolic Glycan Labelling? Q: What is metabolic glycan labelling?

Incorporation of modified sugars into cellular glycans Occurs via normal cellular metabolism Enables covalent tagging of cell surface molecules

IMAGING T CELL FUNCTION Cell labelling strategies Role of the Golgi Q: What role does the Golgi apparatus play in metabolic labelling?

Adds glycan groups to proteins Modified sugars become part of cell surface glycoproteins

IMAGING T CELL FUNCTION Cell labelling strategies Conjugation Concept Q: Why is conjugation important in metabolic labelling?

Allows attachment of probes (fluorophores, drugs, tags) Makes the method multipurpose

IMAGING T CELL FUNCTION Cell labelling strategies Two-Step Glycan Labelling Q: What are the two steps in metabolic glycan labelling

1. Incorporate modified sugar (e.g. Ac₄ManNAz) 2. Attach probe via bioorthogonal click chemistry (DBCO)

IMAGING T CELL FUNCTION Flow Cytometry Q: What is flow cytometry?

Technique to measure physical and chemical properties of cells Cells flow in a fluid stream past a laser

IMAGING T CELL FUNCTION Flow Cytometry What Does Flow Cytometry Measure? Q: What parameters are measured in flow cytometry?

Size → forward scatter Granularity/complexity → side scatter Fluorescence intensity → labelled molecules

IMAGING T CELL FUNCTION Flow Cytometry Fluorescence in Flow Cytometry Q: What is the role of fluorophores in flow cytometry?

Emit light when excited by laser Allow quantitative measurement of labelled cells

IMAGING T CELL FUNCTION Flow Cytometry Identifying Cell Types Q: How can flow cytometry distinguish cell types?

Based on: Size Internal complexity Fluorescence markers

IMAGING T CELL FUNCTION Flow Cytometry Proliferation Dye Principle Q: How do proliferation dyes (CFSE, CTV, CFDA-SE) work?

Label cells with fluorescent dye Dye is split equally during cell division

IMAGING T CELL FUNCTION Flow Cytometry Dye Dilution Mechanism Q: What happens to fluorescence intensity during cell division?

Each division → fluorescence halves Creates distinct peaks for each generation

IMAGING T CELL FUNCTION Flow Cytometry Flow Cytometry Output Q: How does flow cytometry display proliferation data?

Histogram with peaks representing generations Each peak = one round of division

IMAGING T CELL FUNCTION Flow Cytometry Initial Population Signal Q: What does the initial labelled population look like?

Bright, uniform fluorescence

IMAGING T CELL FUNCTION T Cell Labelling Applications Q: Why is metabolic labelling useful for T cell studies?

Enables in vivo tracking of T cells Helps predict therapeutic efficacy

IMAGING T CELL FUNCTION Signal Retention Q: How long can metabolic labelling signals persist

Typically 2–3 days or longer More stable than many non-covalent dyes

IMAGING T CELL FUNCTION Big Picture Q: Why combine metabolic labelling with flow cytometry?

Track cells over time Quantify proliferation and behaviour Analyse immune responses in detail

IMAGING T CELL FUNCTION Key Comparison Q: Why is metabolic labelling preferred over some other methods?

Lower cytotoxicity Stable signal Versatile conjugation options

TME What is the Tumour Microenvironment (TME)? Q: What characterises the tumour microenvironment in this model?

Tumour cells grow rapidly and densely Form heterogeneous regions (e.g. proliferative, hypoxic, necrotic zones)

Tumour Structure in 3D Models Q: Why do tumours develop different regions?

Limited diffusion of oxygen and nutrients Leads to: Outer proliferating cells Inner hypoxic/necrotic core

Visualising the TME Q: How are tumour spheroids and T cells visualised?

Confocal fluorescence microscopy Tumour cells: GFP-labelled (green) T cells: metabolically labelled (red, e.g. Cy5*)

Why Use Metabolic Labelling for T Cells? Q: Why is metabolic glycan labelling used for T cells?

Provides stable, long-term signal retention Enables tracking of T cell infiltration over time

Purpose of T Cell Tracking Q: What is the goal of tracking T cells in the TME?

Measure infiltration into tumours Assess cytotoxic activity (tumour cell killing)

Targeted Immune Response Q: Why is a targeted immune response important in tumour models?

Improves specificity of T cell killing Helps overcome TME barriers and immunosuppression

What is the OT-1 Model? Q: What is the OT-1 transgenic mouse model?

T cells express a specific TCR Recognises OVA-derived peptide (SIINFEKL) on MHC I

OT-1 Antigen Recognition Q: What antigen does OT-1 TCR recognise?

OVA257–264 peptide (SIINFEKL) Presented on MHC class I (H-2Kb)

Why Use OT-1 Model? Q: Why is the OT-1 system useful in tumour studies?

Enables controlled, antigen-specific T cell response Allows precise study of CD8⁺ T cell cytotoxicity

OT-1 T Cell Function Q: What do OT-1 CD8⁺ T cells do in tumour models?

Recognise OVA-expressing tumour cells Infiltrate tumour spheroids Kill target cells

Tracking Targeted Response Q: How can targeted T cell activity be monitored?

Fluorescent tracking of T cells (red) Imaging tumour cells (green) Observe infiltration + tumour cell death over time

Time-Based Analysis Q: What does comparing 1 h vs 24 h imaging show?

Increased T cell infiltration over time Progressive tumour cell killing

TME Barriers to T Cells Q: What challenges do T cells face in the TME?

Physical barriers (dense structure) Immunosuppression Limited penetration into tumour core

Improving T Cell Activity Q: How can T cell activity be enhanced in tumours?

Increase specificity (e.g. OT-1 system) Boost activation to overcome TME suppression

Role of 3D Models Q: Why are tumour spheroids used instead of 2D cultures?

Better mimic real tumour architecture Reflect TME complexity

Big Picture Q: What is the overall aim of these experimental approaches?

Track T cell behaviour in tumours Understand immune-tumour interactions Improve immunotherapy strategies

Summary Q: What key techniques are combined in this study approach?

Metabolic cell labelling Confocal imaging Flow cytometry (optional analysis) Antigen-specific models (OT-1)

week 10 - Imaging T cell function Flashcards

(86 cards)