Mphocytes can not be distinguished by optical microscopy asa result of their similar size ranges and optical properties [52,53]. A sample of splenocytes was collected from the spleen of a sacrificed mouse and purified by lysis and centrifugation. FACS measurements showed that T lymphocytes and B lymphocytes accounted for 29 and 67 of the whole splenocyte population, respectively (Figure S2 in File S1). T lymphocytes were selectively Bromopyruvic acid web labeled in the sample by fluorescent R-phycoerythrin conjugated with anti-CD3e antibody. Anti-CD19 and anti-CD90 antibodies immobilized on a surface via ODN hybridization were recently shown to be able to selectively bind to B and T cells, respectively, [14] and were thus chosen for the functionalization of micropores (Figure 4A). Prior to incubation with cells, the micropore chips were treated with bovine serum albumin (BSA) to minimize non-Cell UKI-1 cost capture by Bio-Functionalized MicroporesFigure 4. Selective capture of B or T lymphocytes from primary splenocyte samples using specific antibody-functionalized micropores. Only the T lymphocytes are fluorescently labeled. A. Schematic illustration of micropore functionalization with antibodies targeting cell surface proteins. B. Transmission and fluorescence microscopy images of cells captured in antibody-functionalized micropores, and stacks of the images. The white dashed circles in the fluorescence images indicate the position of the micropore wall. doi:10.1371/journal.pone.0057717.gspecific adsorption of cells on the surface and immersed in PBS buffer. The chips were placed over an inverted microscope for in situ observation of the micropores (Figure S3 in File S1). 10 mL of splenocyte suspension was loaded from above the zone of each micropore. A few minutes after cell deposition, cells sedimented onto the chip membrane and entered inside the micropore. Movies S1 and S2 show cells passing within the micropores inclose vicinity or in contact with the pore surface. The proximity of flowing cells with the pore surface suggests that biomolecular interactions are prone to be established between the translocating cells and antibody probes. For antibody-functionalized micropores, two different phenomena were observed (Movie S1): individual cells either translocated through or stopped inside 
the pores. Epifluorescence microscopy revealed specific immobilization of T lymphocytes in anti-CD90-Cell Capture by Bio-Functionalized Microporesmodified micropores, and B lymphocytes in anti-CD19-functionalized micropores (Figure 4B). In our experiment 
conditions, we observed that most of the antibody-functionalized pores trapped a cell among the 10 first translocating cells. No non-specific cell capture was observed in our experiments. It is worth to notice that once captured, cells remained immobilized in the micropores for the whole experiment. ODN-modified micropores were used for control experiments to monitor the non-specific cell translocation. In absence of specific interaction, cells translocated through the 10 mm-deep micropores within 1 to 3 seconds (Movie S2). This time range is close to the translocation durations of PS-ncODN measured above using the resistive-pulse technique (1.260.7 s). Furthermore, no cell immobilization inside the control pores was observed (Movie S2). The above results confirm that antibody-functionalized micropores ensure selective capture of well-defined cell types from a complex mixture of cells. In conclusion, we have demonstrated that lo.Mphocytes can not be distinguished by optical microscopy asa result of their similar size ranges and optical properties [52,53]. A sample of splenocytes was collected from the spleen of a sacrificed mouse and purified by lysis and centrifugation. FACS measurements showed that T lymphocytes and B lymphocytes accounted for 29 and 67 of the whole splenocyte population, respectively (Figure S2 in File S1). T lymphocytes were selectively labeled in the sample by fluorescent R-phycoerythrin conjugated with anti-CD3e antibody. Anti-CD19 and anti-CD90 antibodies immobilized on a surface via ODN hybridization were recently shown to be able to selectively bind to B and T cells, respectively, [14] and were thus chosen for the functionalization of micropores (Figure 4A). Prior to incubation with cells, the micropore chips were treated with bovine serum albumin (BSA) to minimize non-Cell Capture by Bio-Functionalized MicroporesFigure 4. Selective capture of B or T lymphocytes from primary splenocyte samples using specific antibody-functionalized micropores. Only the T lymphocytes are fluorescently labeled. A. Schematic illustration of micropore functionalization with antibodies targeting cell surface proteins. B. Transmission and fluorescence microscopy images of cells captured in antibody-functionalized micropores, and stacks of the images. The white dashed circles in the fluorescence images indicate the position of the micropore wall. doi:10.1371/journal.pone.0057717.gspecific adsorption of cells on the surface and immersed in PBS buffer. The chips were placed over an inverted microscope for in situ observation of the micropores (Figure S3 in File S1). 10 mL of splenocyte suspension was loaded from above the zone of each micropore. A few minutes after cell deposition, cells sedimented onto the chip membrane and entered inside the micropore. Movies S1 and S2 show cells passing within the micropores inclose vicinity or in contact with the pore surface. The proximity of flowing cells with the pore surface suggests that biomolecular interactions are prone to be established between the translocating cells and antibody probes. For antibody-functionalized micropores, two different phenomena were observed (Movie S1): individual cells either translocated through or stopped inside the pores. Epifluorescence microscopy revealed specific immobilization of T lymphocytes in anti-CD90-Cell Capture by Bio-Functionalized Microporesmodified micropores, and B lymphocytes in anti-CD19-functionalized micropores (Figure 4B). In our experiment conditions, we observed that most of the antibody-functionalized pores trapped a cell among the 10 first translocating cells. No non-specific cell capture was observed in our experiments. It is worth to notice that once captured, cells remained immobilized in the micropores for the whole experiment. ODN-modified micropores were used for control experiments to monitor the non-specific cell translocation. In absence of specific interaction, cells translocated through the 10 mm-deep micropores within 1 to 3 seconds (Movie S2). This time range is close to the translocation durations of PS-ncODN measured above using the resistive-pulse technique (1.260.7 s). Furthermore, no cell immobilization inside the control pores was observed (Movie S2). The above results confirm that antibody-functionalized micropores ensure selective capture of well-defined cell types from a complex mixture of cells. In conclusion, we have demonstrated that lo.
