Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins David Stepensky."— Presentation transcript:

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

2 Classical pathway of major histocompatibility complex (MHC) class I antigen processing, loading, and presentation Groothuis et al, Immunol Rev 2005

3 Objectives 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 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 Cresswell et al, Immunol Rev 2005 to study the interactions within the MHC I loading complex using fluorescently-tagged components:

4 Fluorescence Resonance Energy Transfer transfer of excited state energy from one fluorophore to another CFP YFP HC Tapasin Excitation & emission spectra CFPYFP HC Tapasin CFP YFP CFP excitationCFP & FRET signals YFP excitationYFP signal %FRET extent of interaction

5 Experimental setup 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 Tapasin Tapasin-YFP Tapasin C95A-YFP controls experimental cell lines HLA- A2.1- CFP HLA- A2.1 T134K -CFP HLA- B44- CFP HLA-A2.1- YFP-CFP HLA- A2.1- CFP

6 FACSAria Violet laser 405 nm CFP (450/40 nm) FRET (530/30 nm) YFP (530/30 nm) Excitation & emission spectra CFP YFP Flow Cytometric Analysis of FRET Objective: to obtain statistically robust measurement of FRET efficiency in the studied cell lines FACS setup: CFPFRET/ YFP Blue laser 488 nm one laser at a time, sequential acquisition of the same sample

7 Negative control Exper. sample Positive control FRET CFP cells YFP FACS-FRET: the raw data FRET CFP positive control experimental sample negative control

8 Tapasin-YFP Tapasin C95A-YFP Tapasin-YFP Tapasin C95A-YFP Tapasin-YFP Tapasin C95A-YFP w/o Tapasin Tapasin Tapasin-YFP Tapasin C95A-YFP HLA- A2.1- CFP HLA- A2.1 T134K -CFP HLA- B44- CFP HLA-A2.1- YFP-CFP HLA- A2.1- CFP FRET/CFP ratio, normalized FACS-FRET: the results controls experimental cell lines FACS-FRET results are consistent with the confocal data both techniques seem to quantify correctly the interaction between the constructs

9 FACS (using the applied setup) Confocal microscopy Acquisition speed high (~10 3 cells/s)low (~10 2 cells/h) Origin of FRET, CFP & YFP signals different cellsthe same cell the whole cellindividual organelles FRET quantification relative valueabsolute value Sorting of cell populations possibleimpossible FRET assessment using FACS or confocal microscope: selected characteristics

10 Dye, Clin Appl Immunol Rev, 2005 He et al, Cytometry Part A, 2003 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 Alternative setups for FACS-FRET

11 FACS Analysis of FRET simple setup combination of FACS with Confocal analysis possibility of cell sorting

12 Cell Sorter Facility Prof. Peter Cresswell and the group Thanks Geoff Lyon Tom Taylor Don Foster


Download ppt "Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins David Stepensky."

Similar presentations


Ads by Google