Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins
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Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins. David Stepensky. Classical pathway of major histocompatibility complex (MHC) class I antigen processing, loading, and presentation. Groothuis et al, Immunol Rev 2005. Objectives.

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Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins

David Stepensky


Classical pathway of major histocompatibility complex (MHC) class I antigen processing, loading, and presentation

Groothuis et al, Immunol Rev 2005


Objectives
Objectives class I antigen processing, loading, and presentation

to study the interactions within the MHC I loading complex using fluorescently-tagged components:

  • the kinetics and sequence of association and dissociation of the loading complex

  • effects of the individual interactions on the loading of the MHC class I molecules with peptides

Cresswell et al, Immunol Rev 2005

Approach

  • generation of fluorescently tagged components of the loading complex

  • investigation of functioning of the tagged proteins

fluorescence-based techniques (FRET, FRAP, etc.)

biochemical techniques


Fluorescence resonance energy transfer
Fluorescence Resonance Energy Transfer class I antigen processing, loading, and presentation

transfer of excited state energy from one fluorophore to another

CFP

HC

Tapasin

CFP

YFP

Excitation & emission spectra

YFP

Tapasin

HC

CFP

YFP

CFP excitationCFP & FRET signals

YFP excitationYFP signal

extent of interaction

%FRET


Experimental setup
Experimental setup class I antigen processing, loading, and presentation

experimental cell lines

controls

  • interaction between MHC class I HC & Tapasin

  • M553 tapasin deficient melanoma

  • stable transfection with:

  • Tapasin-YFP &

  • MHC I heavy chain-CFP

  • multiclonal cell lines

  • interaction (FRET efficiency) was measured using confocal microscope (n=15-20 cells)

FRET, %

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

w/o Tapasin

w/o Tapasin

Tapasin

Tapasin-YFP

Tapasin C95A-YFP

HLA-B44-CFP

HLA-A2.1-

CFP

HLA-A2.1-YFP-CFP

HLA-A2.1-CFP

HLA-A2.1 T134K-CFP


Flow Cytometric Analysis of FRET class I antigen processing, loading, and presentation

Objective:

to obtain statistically robust measurement of FRET efficiency in the studied cell lines

CFP

  • FACSAria

  • Violet laser 405 nm

FACS setup:

Excitation & emission spectra

CFP (450/40 nm)

FRET(530/30 nm)

YFP

  • Blue laser 488 nm

YFP (530/30 nm)

  • one laser at a time, sequential acquisition of the same sample

405

CFP

488

FRET/YFP


FACS-FRET: the raw data class I antigen processing, loading, and presentation

Negative

control

Exper.

sample

FRET

CFP

Positive

control

positive control

experimental sample

negative control

FRET

cells

CFP

YFP


FACS-FRET: the results class I antigen processing, loading, and presentation

  • FACS-FRET results are consistent with the confocal data

  • both techniques seem to quantify correctly the interaction between the constructs

experimental cell lines

controls

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

w/o Tapasin

w/o Tapasin

Tapasin

Tapasin-YFP

Tapasin C95A-YFP

FRET/CFP ratio, normalized

HLA-B44-CFP

HLA-A2.1-

CFP

HLA-A2.1-YFP-CFP

HLA-A2.1-CFP

HLA-A2.1 T134K-CFP


FRET assessment using FACS or confocal microscope: class I antigen processing, loading, and presentation

selected characteristics


Alternative setups for FACS-FRET class I antigen processing, loading, and presentation

He et al, Cytometry Part A, 2003

Dye, Clin Appl Immunol Rev, 2005

  • FACSVantage SE

  • spatial separation of the laser lines

  • optional laser

  • nonstandard mirrors/filters

  • FACSVantage SE

  • laser tuning to 458 nm

  • simultaneous excitation of CFP & YFP

  • nonstandard mirrors/filters


FACS Analysis of FRET class I antigen processing, loading, and presentation

  • simple setup

  • combination of FACS with Confocal analysis

  • possibility of cell sorting


Thanks class I antigen processing, loading, and presentation

Prof. Peter Cresswell and the group

Cell Sorter Facility

Geoff LyonTom TaylorDon Foster


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