Journal: Journal of Biomedical Optics

Article Title: Evaluating integrin activation with time-resolved flow cytometry

doi: 10.1117/1.JBO.23.7.075004

Figure Lengend Snippet: Stage-by-stage depiction of the integrin conformational changes. The U937 cells transfected with a nondesensitizing mutant of the formyl peptide receptor (FPR1 Δ ST ), constitutively expressing target integrin dimer α 4 β 1 , were studied in their: (a) low affinity, resting (bent) conformation. Here, the donor probe (LDV-FITC, 100 nM) was introduced into the cell suspension. This resulted in the rapid binding of the probe to its specific binding site on the α 4 integrin headgroup. As reported previously, 100-nM LDV-FITC concentration was sufficient to fully saturate all low affinity VLA-4 sites and the subsequent inside-out activation did not cause any additional probe binding. (b) The acceptor probe PKH26 is introduced into the cell suspension, where it rapidly partitioned into the plasma membrane. As shown previously, this results in the rapid quenching of the donor fluorescence. (c) The GPCR-induced inside-out integrin activation. The high affinity ligand for FPR1, N-formyl-Met-Leu-Phe-Phe-OH peptide (fMLFF) was added at a saturating concentration. This triggered a series of intracellular signaling events that led to modulation of the VLA-4 integrin ligand-binding affinity and promoted the extended integrin conformation. An increase in the distance of closest approach between FRET donor and acceptor results in the loss of FRET and rapid dequenching of donor emission. (d) The loss of FRET is observed.

Article Snippet: A peptide derivative probe based on a high affinity VLA-4 specific ligand, 4-[(N-2-methylphenyl)ureido]-phenylacetyl- l -leucyl- l –aspatyl- l -valyl- l -prolyl- l -alanyl- l -lysine (LDV peptide), was conjugated to fluorescein isothiocyanate (LDV-FITC, American International Biotechnology, LLC., Richmond, Virginia).

Techniques: Transfection, Mutagenesis, Expressing, Suspension, Binding Assay, Concentration Assay, Activation Assay, Clinical Proteomics, Membrane, Fluorescence, Ligand Binding Assay

Journal: Journal of Biomedical Optics

Article Title: Evaluating integrin activation with time-resolved flow cytometry

doi: 10.1117/1.JBO.23.7.075004

Figure Lengend Snippet: Flow cytometry evaluation of FRET. (a) Dot plot of forward-scatter versus side-scatter light, indicating gated region of cells and (b) fluorescence intensity versus time to depict changes that correlate when cells are labeled and treated for FRET and loss of FRET. Cell populations are time gated to rectify outliers. Gate A : MFI autofluorescence = 4881 and CV = 28 % , gate B : MFI LDV - FITC = 379,900 and CV = 42 % , gate C : MFI FRET = 41,660 with a CV = 55 % , and gate D : MFI Loss of FRET = 48,250 with CV = 56 % . (c) Overlay of histograms of autofluorescence intensities acquired pre-FRET, fluorescence intensity of donor probe before FRET, during FRET, and donor dequenching (integrin activation).

Article Snippet: A peptide derivative probe based on a high affinity VLA-4 specific ligand, 4-[(N-2-methylphenyl)ureido]-phenylacetyl- l -leucyl- l –aspatyl- l -valyl- l -prolyl- l -alanyl- l -lysine (LDV peptide), was conjugated to fluorescein isothiocyanate (LDV-FITC, American International Biotechnology, LLC., Richmond, Virginia).

Techniques: Flow Cytometry, Fluorescence, Labeling, Activation Assay

Journal: Journal of Biomedical Optics

Article Title: Evaluating integrin activation with time-resolved flow cytometry

doi: 10.1117/1.JBO.23.7.075004

Figure Lengend Snippet: Average fluorescence lifetime values measured per integrin conformational event. Fluorescence lifetime values were obtained for LDV-FITC donor probe, quenching of the donor probe, and dequenching of the donor probe. (a) The average LDV-FITC donor lifetime = 4.3 ± 0.2 ns , (b) the average FRET lifetime = 2.7 ± 0.5 ns , and (c) average loss of FRET lifetime = 3.2 ± 0.5 ns .

Article Snippet: A peptide derivative probe based on a high affinity VLA-4 specific ligand, 4-[(N-2-methylphenyl)ureido]-phenylacetyl- l -leucyl- l –aspatyl- l -valyl- l -prolyl- l -alanyl- l -lysine (LDV peptide), was conjugated to fluorescein isothiocyanate (LDV-FITC, American International Biotechnology, LLC., Richmond, Virginia).

Techniques: Fluorescence

Journal: Journal of Biomedical Optics

Article Title: Evaluating integrin activation with time-resolved flow cytometry

doi: 10.1117/1.JBO.23.7.075004

Figure Lengend Snippet: Graphical representation of the FRET experiment in phasor plots. After calibration, the demodulation depth and phase values are plotted on phasor graphs. Plots follow order in which the analysis was performed; donor fluorescence lifetime, showing a consensus for a single lifetime value. FRET (FITC donor quenched by acceptor PKH26) with the population moving inside the semicircle indicative of multiple lifetime values and loss of FRET with bimodal populations present, showing the nonresponsive population, partial recovery population, and recovered population.

Article Snippet: A peptide derivative probe based on a high affinity VLA-4 specific ligand, 4-[(N-2-methylphenyl)ureido]-phenylacetyl- l -leucyl- l –aspatyl- l -valyl- l -prolyl- l -alanyl- l -lysine (LDV peptide), was conjugated to fluorescein isothiocyanate (LDV-FITC, American International Biotechnology, LLC., Richmond, Virginia).

Techniques: Fluorescence

Journal: Journal of Biomedical Optics

Article Title: Evaluating integrin activation with time-resolved flow cytometry

doi: 10.1117/1.JBO.23.7.075004

Figure Lengend Snippet: Phasor overlay showing LDV-FITC, FRET, and loss of FRET population distributions with trajectory. Trajectory tracks the donor’s decay kinetics as it undergoes FRET, followed by its recovery. The trajectory allows evaluation of FRET efficiencies by observing the order of events in the phasor space. The FRET efficiency was calculated to be 37%. In addition, the donor recovery post-FRET was calculated at 26%. This plot is substantially quantitative as it adds a spatial component to compliment the temporal FRET evaluation.

Article Snippet: A peptide derivative probe based on a high affinity VLA-4 specific ligand, 4-[(N-2-methylphenyl)ureido]-phenylacetyl- l -leucyl- l –aspatyl- l -valyl- l -prolyl- l -alanyl- l -lysine (LDV peptide), was conjugated to fluorescein isothiocyanate (LDV-FITC, American International Biotechnology, LLC., Richmond, Virginia).

Techniques: