Fig. 4: On-chip screening for infected MDA-MB-231 cells after bacteria were labeled with DiO. | Nature Communications

Fig. 4: On-chip screening for infected MDA-MB-231 cells after bacteria were labeled with DiO.

From: A Mechanophenotyping chip for high-throughput detection of metastatic bacteria-infected circulating tumor cells

Fig. 4: On-chip screening for infected MDA-MB-231 cells after bacteria were labeled with DiO.

a Schematic showing experimental procedure. Native MDA-MB-231 cells were stained with Hoechst and bacteria were labeled with DiO before infection. A mixture of native and infected cells was added to LesM chip under the optimal conditions of a cell concentration of 1 × 106 cells·mL-1, a flow velocity of 5 μL·min-1, and a deformation channel width of 6 μm. Icons were created in BioRender. Luo, W. (https://BioRender.com/l1e6nxj). b, c Representative micrographs showing blocked-cell and squeezed-cell ROIs, with blue fluorescence indicating Hoechst staining and green, DiO-labeled bacteria. Scale bars, 100 μm. Cells are MDA-MB-231 cells infected with (b) S. xylosus or (c) L. animalis. d–f Data showing fluorescence from 2 ROIs after MDA-MB-231 cells, some invaded by S. xylosus and some not, were added to the chip. Violin plots of fluorescence from (d) blocked-cell ROIs, (e) squeezed-cell ROIs, and (f) density plot. Each dot represents one cell, with n = 800 (biologically independent). Lower boundary of the shaded regions represents the threshold of meaningful fluorescence; below this boundary, any fluorescence is considered background fluorescence, and numbers 1-5 represent 5 independent experiments under the same conditions. g ROC curves of 5 replicated tests for the discrimination of S. xylosus–infected MDA-MB-231 cells by fluorescence from squeezed-cell ROIs. h–j Data showing fluorescence from 2 ROIs after MDA-MB-231 cells, some invaded by L. animalis and some not, were added to the chip. Violin plots of fluorescence from (h) blocked-cell ROIs, (i) squeezed-cell ROIs, and (j) Density plot. k ROC curves of 5 replicated tests for the discrimination of L. animalis–infected MDA-MB-231 cells by fluorescence from squeezed-cell ROIs. AUC area under the ROC curve, bac bacteria, BF bright-field microscopy, DiO 3,3’-dioctadecyloxacarbocyanine perchlorate, FL fluorescence intensity, ROC receiver operating characteristic, ROI region of interest.

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