Journal: eLife

Article Title: Redox regulation of PTPN22 affects the severity of T-cell-dependent autoimmune inflammation

doi: 10.7554/eLife.74549

Figure Lengend Snippet: ( A ) Gene expression of PTPN22 in splenocytes shown as fold change over WT. ( B ) Immunoblot analysis of PTPN22 expression in sorted CD4 T cells either unstimulated or activated in vitro with 1 µg/ml anti-CD3/CD28 for 24 hr. Each lane shows cells from a distinct mouse, and relative expression to the right was calculated by normalizing to loading control (actin). ( C, D ) Analysis of T cell populations in naïve mice via flow cytometry. ( C ) Analysis of double negative (DN), double positive (DP), and single positive CD4 and CD8 thymic populations in 10-week-old littermate mice as well as expression of TCRb, CD5, and CD69 on various subsets. ( D ) Frequencies of activated (CD44+/ CD69+) and regulatory (FOXP3+) CD4+ T cells in thymus and secondary lymphoid organs. ( E ) Intracellular calcium measurement in CD4 T cells at baseline, after stimulation with anti-CD3, and ionomycin to achieve maximum Ca 2+ influx via staining with Fluo4 and FuraRed. Shown is a representative image of the change in ratio of fluo4 to FuraRed expression over time (blue: wild-type; red: PTPN22 C129S ). Quantification to the right shows slope value that describes how fast the peak of Ca 2+ influx is reached . ( F ) Proliferation of CD4+ T cells as assessed by CellTrace Violet dilution after 96 hr in vitro stimulation with 1 µg/ml anti-CD3/CD28. Proliferation Index is the total number of divisions divided by the number of cells that went into division. Representative proliferation peaks on the left (clear: wild-type; black: PTPN22 C129S ) and quantification on the right (ctrl refers to unstimulated samples). ( G, H ) Ex vivo-stimulated CD4 T cells were assessed for CD69 expression/IFNγ production after stimulation with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin, respectively. Representative gating shown. Error bars represent mean ± SEM. Figure 5—source data 1. Uncropped Western blot images showing PTPN22 expression in CD4+ T cells. Figure 5—source data 2. Raw images of PTPN22 expression in CD4+ T cells.

Article Snippet: Proliferation of CD4+ T cells purified by negative sorting (Thermo, 11416D) was assessed using the CellTrace Proliferation Kit (Thermo, C34554) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, In Vitro, Flow Cytometry, Staining, Ex Vivo

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A high-throughput technique to map cell images to cell positions using a 3D imaging flow cytometer

doi: 10.1073/pnas.2118068119

Figure Lengend Snippet: Human cancer cell experiment. MCF-7 and HeLa cells were stained with CFSE and CellTrace yellow, respectively, and HEK-293 were unstained. ( A ) Representative section of the matched sequences from the IFC and CPP readouts. The number between the adjacent marker beads shows the number of cells in between. When the number of cells in two sequences matches, we can unambiguously match the cell with its 3D images. [* indicates section that is expanded in B ( i ) and ( ii )]. ( B ) A representative section (in the box in A ) of cells ( i ) on the CPP membrane and ( ii ) having their 3D SSC images and fluorescent images (in yellow and green from CellTrace Yellow and CFSE, respectively) as well as 2D transmission images. Notice that cell 2 appears to be a doublet. Cell 11 shows a doublet in the scattering and fluorescent images, but the 2D transmission image can resolve the doublet from the perspective. CFSE, Cell Proliferation Kit (Ex/Em, 488/517); yellow, CellTrace yellow proliferation kit (Ex/Em, 546/579); SSC, side scattering (90 degrees); Tm, transmission image. (Scale bar, 10 µm.)

Article Snippet: MCF-7 cells and HeLa cells were fluorescently stained with the carboxyfluorescein succinimidyl ester (CFSE) (excitation/emission [Ex/Em] wavelengths: 492/517 nm, Thermo Fisher) and the CellTrace yellow proliferation kit (Ex/Em 546/579 nm, Thermo Fisher), respectively.

Techniques: Staining, Marker, Membrane, Transmission Assay