Research ArticleIMMUNOLOGY

Microfluidics-based super-resolution microscopy enables nanoscopic characterization of blood stem cell rolling

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Science Advances  18 Jul 2018:
Vol. 4, no. 7, eaat5304
DOI: 10.1126/sciadv.aat5304
  • Fig. 1 Microfluidics-based in vitro cell-rolling assay for characterizing HSPC rolling.

    (A) Experimental configuration of the microfluidics SR microscopy platform. (B) Transmitted light microscopy images of KG1a cells rolling on E-selectin. The surface density of the E-selectin was 3.6 molecules μm−2. (C) Wall shear stress–dependent rolling velocity of KG1a cells (blue) and the number of bound cells to the E-selectin surface (red). Error bars indicate SDs of six different fields of view. (D) E-selectin density–dependent rolling velocity of KG1a cells (blue) and the number of bound cells to the E-selectin surface (red). Error bars indicate SDs of six different fields of view. CCD, charge-coupled device; EMCCD, electron multiplying CCD.

  • Fig. 2 SR localization microscopy imaging of CD44 on KG1a cells.

    (A) Confocal fluorescence image of CD44 on a KG1a cell that was fixed and immunolabeled for CD44 using the 515 antibody followed by AF-647–conjugated secondary antibodies. (B) Confocal fluorescence image of CD44 on a KG1a cell that was fixed and immunolabeled by AF-647 dye in the microfluidic chamber after the rolling of the cell on E-selectin. (C) SR image of CD44 on a KG1a cell that was fixed and immunolabeled for CD44 using the 515 antibody followed by AF-647–conjugated secondary antibodies. The inset shows an enlarged view of the yellow region. (D) SR images of CD44 on a KG1a cell that was fixed and immunolabeled by AF-647 dye in the microfluidic chamber after the rolling of the cell on E-selectin. (E) 3D SR image of CD44 on a KG1a cell that was fixed and immunolabeled by AF-647 dye in suspension. The insets show enlarged views of the blue regions. (F) 3D SR images of CD44 on a KG1a cell that was fixed and immunolabeled by AF-647 dye in the microfluidic chamber after the rolling of the cell on E-selectin. The inset shows an enlarged view of the blue region. (G) Frequency histogram of the width of the CD44 clusters observed in the control cells. (H) Frequency histogram of the width of the CD44 clusters observed in the rolled KG1a cells.

  • Fig. 3 Two-color SR images of CD44 and actin on KG1a cells.

    (A) SR image of CD44 (cyan) and actin cytoskeleton (yellow) on a KG1a cell that was fixed and immunolabeled by AF-647 (CD44) and labeled by AF-488 dye–conjugated phalloidin (actin). The insets show enlarged views of the yellow regions. (B) SR image of CD44 (cyan) and actin cytoskeleton (yellow) on a KG1a cell that was fixed and fluorescently labeled in the microfluidic chamber after the rolling of the cell on E-selectin. The inset shows enlarged view of the yellow region.

  • Fig. 4 Two-color SR images of CD44 and lipid rafts on KG1a cells.

    (A) SR image of CD44 (cyan) and lipid rafts (yellow) on a KG1a cell that was fixed and immunolabeled by AF-647 (CD44) and labeled by AF-488–CtxB subunit (lipid rafts). The insets show enlarged views of the yellow regions. (B) SR image of CD44 (cyan) and lipid rafts (yellow) on a KG1a cell that was fixed and fluorescently labeled in the microfluidic chamber after the rolling of the cell on E-selectin. The inset shows enlarged view of the yellow region. (C) Mean cluster sizes of lipid rafts on control and rolled cells on E-selectins. Error bars indicate SDs of the cluster sizes obtained from 12 cells for each condition.

  • Fig. 5 Effect of lipid rafts on the nanoscale reorganization of CD44.

    SR image of (A) CD44 on a methyl-β-cyclodextrin (MβCD)–treated KG1a cell that was fixed and immunolabeled by AF-647 dye in a suspension and (B) CD44 on an MβCD-treated KG1a cell that was fixed and fluorescently labeled in the microfluidic chamber after the rolling of the cell on E-selectin. Two-color SR images of (C) CD44 (cyan) and actin cytoskeleton (yellow) on an MβCD-treated KG1a cell that was fixed and immunolabeled by AF-647 dye in a suspension and (D) CD44 (cyan) and actin cytoskeleton (yellow) on an MβCD-treated KG1a cell that was fixed and fluorescently labeled in the microfluidic chamber after the rolling of the cell on E-selectin. The insets show enlarged view of the yellow regions. (E) Mean cluster sizes of CD44 on control cells, MβCD-treated cells, and MβCD-treated cells after rolling over E-selectin. Error bars indicate SDs of the cluster sizes obtained from 25, 19, and 12 cells for control, MβCD-treated, and MβCD-treated rolled cells, respectively.

  • Fig. 6 Effect of nanoscopic clustering of CD44 on the macroscopic rolling behavior of KG1a cells on E-selectin.

    Transmitted light microscopy images of (A) control and (B) MβCD-treated KG1a cells rolling on E-selectin. The surface density of E-selectin was 2.7 molecules μm−2. (C) Mean rolling velocities of control and MβCD-treated cells. Error bars indicate SDs of six different fields of view. (D) Rolling time dependence of the number of control (black) and MβCD-treated (blue) KG1a cells tethered and rolled on E-selectin. The number is normalized by the number of cells in the first time point. Error bars indicate SDs obtained from two to four independent experiments.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/7/eaat5304/DC1

    Fig. S1. The multistep paradigm of cell migration highlighting the main interactions that take place between the blood stem cell in flow and the endothelial cells lining the blood vessels of the bone marrow.

    Fig. S2. Surface density of E-selectin on the protein A–deposited microfluidic chamber determined by immunofluorescence imaging.

    Fig. S3. Flow cytometric analysis to determine the binding specificity of ligand-specific antibodies to CD44 on KG1a cells.

    Fig. S4. Localization precisions of the SR localization microscopy experiments of CD44 on KG1a cells.

    Fig. S5. Examples of the reconstructed SR images of CD44 on KG1a cells.

    Fig. S6. SR images of CD44 on KG1a cells.

    Fig. S7. Cluster analysis of the nanoscale architecture of lipid rafts on KG1a cells.

    Fig. S8. Examples of the reconstructed SR images of CD44 on MβCD-treated KG1a cells.

    Fig. S9. Cluster analysis of the nanoscale architecture of CD44 on KG1a cells.

    Fig. S10. Expression of CD44 on untreated and MβCD-treated KG1a cells was determined by flow cytometry.

    Fig. S11. Depth of the field in the SR localization microscopy imaging experiments with HILO configuration.

    Movie S1. Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 0.25 dyne cm−2.

    Movie S2. Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 0.5 dyne cm−2.

    Movie S3. Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 1.0 dyne cm−2.

    Movie S4. Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 2.0 dyne cm−2.

    Movie S5. Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 4.0 dyne cm−2.

    Movie S6. Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber in the presence of EDTA (10 mM) at the shear stress of 1.0 dyne cm−2.

    Movie S7. Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 1.0 dyne cm−2.

    Movie S8. Time-lapse transmitted light microscopy images of MβCD-treated KG1a cells perfused into the microfluidic chamber at the shear stress of 1.0 dyne cm−2.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. The multistep paradigm of cell migration highlighting the main interactions that take place between the blood stem cell in flow and the endothelial cells lining the blood vessels of the bone marrow.
    • Fig. S2. Surface density of E-selectin on the protein A–deposited microfluidic chamber determined by immunofluorescence imaging.
    • Fig. S3. Flow cytometric analysis to determine the binding specificity of ligand-specific antibodies to CD44 on KG1a cells.
    • Fig. S4. Localization precisions of the SR localization microscopy experiments of CD44 on KG1a cells.
    • Fig. S5. Examples of the reconstructed SR images of CD44 on KG1a cells.
    • Fig. S6. SR images of CD44 on KG1a cells.
    • Fig. S7. Cluster analysis of the nanoscale architecture of lipid rafts on KG1a cells.
    • Fig. S8. Examples of the reconstructed SR images of CD44 on MβCD-treated KG1a cells.
    • Fig. S9. Cluster analysis of the nanoscale architecture of CD44 on KG1a cells.
    • Fig. S10. Expression of CD44 on untreated and MβCD-treated KG1a cells was determined by flow cytometry.
    • Fig. S11. Depth of the field in the SR localization microscopy imaging experiments with HILO configuration.
    • Legends for movies S1 to S8

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 0.25 dyne cm−2.
    • Movie S2 (.mp4 format). Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 0.5 dyne cm−2.
    • Movie S3 (.mp4 format). Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 1.0 dyne cm−2.
    • Movie S4 (.mp4 format). Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 2.0 dyne cm−2.
    • Movie S5 (.mp4 format). Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 4.0 dyne cm−2.
    • Movie S6 (.mp4 format). Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber in the presence of EDTA (10 mM) at the shear stress of 1.0 dyne cm−2.
    • Movie S7 (.mp4 format). Time-lapse transmitted light microscopy images of KG1a cells perfused into the microfluidic chamber at the shear stress of 1.0 dyne cm−2.
    • Movie S8 (.mp4 format). Time-lapse transmitted light microscopy images of MβCD-treated KG1a cells perfused into the microfluidic chamber at the shear stress of 1.0 dyne cm−2.

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