Journal: Cancers

Article Title: Non-Expressed Donor KIR3DL1 Alleles May Represent a Risk Factor for Relapse after T-Replete Haploidentical Hematopoietic Stem Cell Transplantation

doi: 10.3390/cancers15102754

Figure Lengend Snippet: Intracellular localization of KIR3DL1*019. Flow cytometry analysis of Jurkat cell line after stable transfection with KIR3DL1*002, *004, *019, *019 L86S , *019 S182P , and *019 L86S+S182P chimeric constructs containing eGFP. Only cells positive for eGFP fluorescence were examined. ( a ) Specific Z27 3.7 anti-KIR3DL1 mAb (blue peak; Beckman Coulter) with isotype MOPC-21 IgG control mAb (green peaks; Sony) were used in extracellular staining; ( c ) specific 177,407 anti-KIR3DL1 mAb (blue peak; R&D Systems) with isotype 11,711 IgG control (green peaks; R&D Systems) were used in intracellular staining. Fluorescent microscopy of Jurkat cells stably transfected with KIR3DL1*002, *004, *019, *019 L86S , *019 S182P , and *019 L86S+S182P chimeric constructs containing eGFP. F41-1E3 anti-HLA class I mAb (EFS Nantes) was used in ( b ) extracellular and ( d ) intracellular transfectant staining. Magnification 100×. Specific Z27 3.7 (Beckman Coulter) and 177,407 (R&D Systems) anti-KIR3DL1 mAbs were used in extracellular and intracellular staining, respectively. Merged images show co-localization of KIR3DL1-eGFP, HLA class I delimiting plasma membrane and KIR3DL1 in extracellular and intracellular staining.

Article Snippet: Intracellular labeling was performed on cells permeabilized with BD Cytofix/Cytoperm solution (Becton Dickinson, Franklin Lakes, NJ, USA) using the AF647-conjugated anti-KIR3DL1 mAb (clone 177407; R&D Systems), the corresponding IgG1 isotype control (clone 11711; R&D Systems), the AF555-conjugated anti-HLA class I mAb (clone F41-1E3), and the corresponding AF555-IgG1 isotype control (clone MOPC-21).

Techniques: Flow Cytometry, Stable Transfection, Construct, Fluorescence, Control, Staining, Microscopy, Transfection, Clinical Proteomics, Membrane

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Confirmed chemical structures of cyanine fluorophores from the Cy‐, Alexa Fluor and the AF‐series . For Cy3, Cy5, Sulfo‐Cy3, Sulfo‐Cy5 all structures were obtained from the literature.[ , , ] The structures of AF555 and Alexa Fluor 555 were determined by NMR and mass spectrometry in this manuscript. For AF647, AFD647 and Alexa Fluor 647, we confirmed the published structures (AF(D)647 in, Alexa Fluor 647 structure in ) by mass spectrometry. Please note that the AF‐fluorophore homologues of Cy5 are available in two distinct versions called AFD647 (n=1) and AF647 (n=2).

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques: Mass Spectrometry

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Spectroscopic characterization of bulk solutions of free green and red cyanine fluorophores. (a) Absorbance and emission spectra of Alexa Fluor 555 and AF555 in comparison to Cy3, Sulfo‐Cy3 and Alexa Fluor 546 (left) show red‐shifted spectra for increased number of SO 3 ‐ groups and difference in spectral shape compared to the rhodamine‐derivative Alexa Fluor 546. Absorbance and emission spectra of Alexa Fluor 647, AFD647 and AF647 in comparison to Cy5, Sulfo‐Cy5 and Atto647 N (left) show red‐shifted spectra for increased number of SO 3 ‐ groups with small difference in spectral shape compared to Atto647 N. (b) Lifetime (left) and time‐resolved anisotropy measurements (right) of free Fluor Alexa 555 (lighter green) and AF555 (darker green) at 100 nM concentration. (c) Lifetime (left) and time‐resolved anisotropy measurements (right) of free Fluor Alexa 647 (lighter red) and AFD647 (darker red) at 100 nM concentration. (d) Integrated intensity versus absorbance at 510 nm at five different concentrations (squares) for Alexa Fluor 555 (top) and AF555 (bottom) with absorbance‐corrected curve fit (solid line, see methods). (e) Integrated intensity versus absorbance at 610 nm at five different concentrations (squares) for Alexa Fluor 647 (top) and AF647 (bottom) with absorbance‐corrected curve fit as in (c).

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques: Concentration Assay

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Photophysical and chemical parameters of Alexa Fluor 555/647 and AF555/AF(D)647.

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques:

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Mass spectrometry‐based structure elucidation . Fragmentation mass spectra of the different fluorophores (a) Alexa Fluor 555 and (b) AF555. Mass range was set to 200–1050 m/z. Mass accuracy was 0.23±0.09 ppm. Each fragmentation spectrum includes the predicted or confirmed structure. Dashed lines represent fragmentation events with resulting fragment masses or empirical formulas. M, molecular ion. Red crosses indicate fragment masses that could not be assigned, green ticks for mass fragments that are compatible with the proposed molecular structures.

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques: Mass Spectrometry

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Structure determination of Alexa Fluor 555 via NMR. (a) Based on mass spectrometry we were able to propose two possible Alexa Fluor 555 structures, from which the left structure was confirmed by NMR spectroscopy. (b) Region from the 1 H, 13 C‐HMBC spectrum showing long range correlation via two or three bonds, proving that the H10 and H31 protons are close to C2, and H21 to C2′ (indicated by blue arrows in the structures in panel (a)). From the characteristic 1 H chemical shifts, it also becomes clear that both alkyl sulfonate sidechains are connected to a nitrogen atom (H10 & H21 at 4.2–4.3 ppm), unlike the maleimide sidechain (H31 at 2.1–2.4 ppm), which rules out the alternative structure for Alexa Fluor 555. In addition, 1 H, 1 H though space correlations from NOESY spectra are in complete agreement with the confirmed structure (green arrows), but are widely incompatible with the alternative structure (red arrows).

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques: Mass Spectrometry, Spectroscopy

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Structural analysis of AF555 by NMR . (a) Confirmed and refined structure of AF555 (left) and previously published structure (right, discarded). (b) Region from the 1 H, 13 C‐HMBC spectrum containing long‐range correlations via two or three bonds. The cross‐peaks clearly show that both alkyl‐sulfonate sidechains (with their terminal CH 2 groups H10 and H21) are linked to the same half of the cyanine core (C2′), while the maleimide sidechain is connected to the other half (correlation between H31 and C2). These correlations are depicted by the blue (possible) and red (impossible) arrows in the structures (a). In addition, experimentally observed through‐space correlations from NOESY spectra further confirm this side‐chain arrangement (green lines, thickness correlates with intensity).

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques:

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: smFRET measurement comparison of Alexa Fluor 555 – Alexa Fluor 647 and AF555 – AFD647: (a) Overlayed crystal structures of MalE in apo state (gray, PDB 1omp) and holo state (green, PDB 1anf). Residues 4–103 are aligned and labeled residues are marked with spheres (PyMol). The FRET pairs 36–352, 87–186, and 85–352 are indicated with lines for apo (solid) and holo state (dashed). (b) Representative FRET efficiency vs. stoichiometry plots (ES‐plots) for MalE variant 87–186 labeled with Alexa Fluor 555/Alexa Fluor 647 (left) and AF555/AFD647 (right) to show data quality and ratio of double labeled donor‐acceptor pairs. (c) FRET efficiency histograms (uncorrected, raw FRET values) for the three FRET variants labeled with the Alexa Fluor and AF pair are fitted with a Gaussian fit function with mean μ E and s E of the fit. The distributions show similar FRET efficiency values for all variants in apo state (top) and holo state with 1 mM maltose (bottom). Please note that (a) shows the apo and holo conformation of the protein with insufficient detail to see that MalE85‐352 has no change of inter‐dye distance upon maltose binding.

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques: Labeling, Variant Assay, Binding Assay

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Characterization of anisotropy and lifetime decays of cyanine dyes on MalE: (a) Lifetime (top) and time‐resolved anisotropy measurements (bottom) of Alexa Fluor 555 (lighter green) and AF555 (darker green) labelled at residues 186 (left) and 352 (right) in the ligand‐free state of MalE. (b) Lifetime (top) and time‐resolved anisotropy measurements (bottom) of Alexa Fluor 647 (lighter red) and AF647 (darker red) labelled at residues 186 (left) and 352 (right) in the ligand‐free state of MalE.

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques:

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Average rotational anisotropy decay calculated from MD simulations of fluorophore‐labelled MalE . Mutants A186C and S352C were combined with fluorophores AF555 and Alexa Fluor 555, and multiple (∼20) simulations of 20 ns length were performed per protein‐dye system. The curves depict the average of the anisotropy decay r ( t ) over these simulations. Time origins for the averaging per simulation are separated by 6 ps.

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques:

Journal: Chemphyschem

Article Title: Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **

doi: 10.1002/cphc.202000935

Figure Lengend Snippet: Geometric Parameters for in silico predictions of FRET labels using the FRET‐restrained positioning system.

Article Snippet: The maleimide derivatives of the dyes Alexa Fluor 555 (ThermoFischer Scientific, A20346, 5*1 mg) and AF555 (Jena Bioscience, APC‐007, 2*5 mg) were dissolved in 0.6 mL DMSO‐d6 (99.96 % 2 H, Eurisotope, St‐Aubin, France).

Techniques: In Silico