c2 plus laser scanning confocal head  (Nikon)

Journal: Nature Communications

Article Title: Supramolecular aptamer nano-constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells

doi: 10.1038/s41467-018-02929-2

Figure Lengend Snippet: Confocal microscopy and flow cytometry analysis of Atto647N-trCLN3-L4 internalization. a , b Confocal images of NCI-H1838 cells incubated with Atto647N-labeled trCLN3-L4 (A647N- 3 ) nanoconstructs at 37 °C ( a : unmerged; red; c3 and b : overlay; c1 + c2 + c3). c , d NCI-H1838 cells incubated with Atto647N- 3 at 4 °C ( c : unmerged; red; c3 and d : overlay; c1 + c2 + c3). Arrow: Alexa488-WGA membrane stain (green) shows colocalization with Atto647N- 3 (red). e , f NCI-H1838 cells treated with Atto647N.mut- 3 at 37 °C ( e : unmerged; red; c3 and f : overlay; c1 + c2 + c3). g , h NCI-H1838 cells treated with Atto647N-trCLN3 w/oL4 (without lipid modification) at 37 °C ( g : unmerged; red; c3 and h : overlay; c1 + c2 + c3). Cells were membrane stained with Alexa488-WGA (green; c2), nuclei were stained with Hoechst 33342 (blue; c1) and analyzed for Atto647N- 3 uptake (red; c3). Scale bars: a – h 50 µm. i FACS histograms for cells treated with Atto647N- 3 at 37 °C (green shadow) showed a significant shift in Atto647N fluorescence intensity compared to cells treated with Atto647N- 3 at 4 °C (red solid line), suggesting an endocytotic internalization pathway. A minimal shift in Atto647 fluorescence intensity was observed for cells treated with either a scrambled aptamer Atto647N.mut- 3 (blue solid line) or with Atto647N-trCLN3 w/oL4 (black dotted line) at 37 °C compared to untreated cells (gray shadow), indicating a marginal internalization presumably due to non-specific binding or lack of lipidation

Article Snippet: Finally, the 96-well plate was mounted with a multi-well plate holder and the confocal imaging of the fixed cells was performed by using a NikonTi-E Eclipse inverted confocal laser-scanning microscope equipped with a 60× Plan Apo VC Oil-immersion DIC N2 objective, a Nikon C2 plus confocal laser scan head and a pinhole of 1.2 airy unit (30 μm).

Techniques: Confocal Microscopy, Flow Cytometry, Incubation, Labeling, Membrane, Staining, Modification, Fluorescence, Binding Assay

Journal: Nature Communications

Article Title: Supramolecular aptamer nano-constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells

doi: 10.1038/s41467-018-02929-2

Figure Lengend Snippet: Cellular uptake of dual-labeled HyApNc consisting of A550- 4 and A647N- 3 motifs. a – c Confocal fluorescence images of H1838 cells treated with the HyApNc consisting of Atto550- DxR -L4 motif (A550- 4 ) and Atto647N-trCLN3-L4 (A647N- 3 ) motifs in 1:1 ratio. Both A647N- 3 ( a : c2: red) and A550- 4 ( b : c3: magenta) fluorescence were observed from the cytosol including a FRET-mediated Atto647N signal ( c : c4: cyan). d Calculated FRET signal from reconstructed FRET images ( d : white) indicate the intracellular integrity of the functional nanoconstruct HyApNc. e , f Overlay images of cells incubated with HyApNc ( e : A647N- 3 + A550- 4 ), and HyApNc.mut ( f : A647N.mut- 3 + A550- 4 ) as a negative control with Atto647N-labeled mutant trCLN3.mut-L4 motif. The complete overlay sets for e and f are shown in Supplementary Fig. . Aptamer constructs were incubated at 37 °C for 2 h, followed by membrane staining with Alexa488-WGA (green), and nuclei staining with Hoechst 33342 (blue). Scale bar: a – f 50 µm

Article Snippet: Finally, the 96-well plate was mounted with a multi-well plate holder and the confocal imaging of the fixed cells was performed by using a NikonTi-E Eclipse inverted confocal laser-scanning microscope equipped with a 60× Plan Apo VC Oil-immersion DIC N2 objective, a Nikon C2 plus confocal laser scan head and a pinhole of 1.2 airy unit (30 μm).

Techniques: Labeling, Fluorescence, Functional Assay, Incubation, Negative Control, Mutagenesis, Construct, Membrane, Staining

Journal: Nature Communications

Article Title: Supramolecular aptamer nano-constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells

doi: 10.1038/s41467-018-02929-2

Figure Lengend Snippet: Confocal microscopy and flow cytometry analysis of the HyApNc-mediated doxorubicin uptake. a – c Confocal image of intracellular distribution of DxR released (yellow signal) from the DxR -loaded HyApNc nanoconstructs in the H1838 cells incubated with free DxR ( a : nuclei staining with Hoechst 33342; blue; c1, b : DxR staining; yellow; c2, c : overlay; c1 + c2). d – f Cells incubated with HyApNc- DxR not exposed to UV irradiation ( d : Hoechst 33342; blue; c1, e : DxR staining; yellow; c2, f : overlay; c1 + c2). g – i Cells incubated with HyApNc- DxR exposed to UV light, λ = 365 nm, 350 mW cm −2 ( g : Hoechst 33342; blue; c1, h : DxR staining; yellow; c2, i : overlay; c1 + c2). j – l Cells treated with HyApNc w/oAz - DxR without UV irradiation ( j : Hoechst 33342; blue; c1, k : DxR staining; yellow; c2, l : overlay; c1 + c2). m – o Cells treated with HyApNc w/oAz - DxR exposed to UV light ( λ = 365 nm, 350 mW cm −2 ) ( m : Hoechst 33342; blue; c1, n : DxR staining; yellow; c2, o : overlay; c1 + c2). Blue (c1) and yellow (c2) signals show the fluorescence of Hoechst 33342 and DxR staining, respectively. The overlay (c1 + c2) shows colocalization of Hoechst 33342 and DxR . An increase in nuclear accumulation of DxR upon light triggering was observed only for the photoactivated nanoconstruct. Scale bar: a – o 50 µm. p Flow cytometry histogram showing quantitative comparison of DxR accumulation in H1838 cells after incubation with free DxR (orange shadow), mutant non-targeted nanoconstructs HyApNc.mut- DxR (magenta solid), targeted nanoconstructs HyApNc- DxR without UV (purple solid), or with UV irradiation (red dotted). q Flow cytometry histogram showing DxR accumulation in H1838 cells after incubation with HyApNc w/oAz - DxR without UV (blue solid) or with UV irradiation (red dotted) at 37 °C for 2 h. The concentration of DxR either in free form or its equivalent in complex form in the cell culture was fixed at 8 µM. Untreated cells are shown in shadow (gray). The numbers in bracket of the legends are the geometric mean of the corresponding peaks

Article Snippet: Finally, the 96-well plate was mounted with a multi-well plate holder and the confocal imaging of the fixed cells was performed by using a NikonTi-E Eclipse inverted confocal laser-scanning microscope equipped with a 60× Plan Apo VC Oil-immersion DIC N2 objective, a Nikon C2 plus confocal laser scan head and a pinhole of 1.2 airy unit (30 μm).

Techniques: Confocal Microscopy, Flow Cytometry, Incubation, Staining, Irradiation, Fluorescence, Comparison, Mutagenesis, Concentration Assay, Cell Culture