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CYTOA_22075_sm_SupFig1.tiff1520KSupplemental Figure 1: Comparison of total DNA content measurement of stimulated T cells by fluorescence cytometry and mass cytometry. (A) Comparison of the same sample of stimulated T cells by Hoechst 3342 detected with fluorescence cytometry and mass cytometry. (B) DNA staining of the same cells subdivided by cell cycle phase. For mass cytometric analysis cells were gated as described above; fluorescence cytometry gating was performed on the basis of Hoechst, pyronin Y, and IdU staining.
CYTOA_22075_sm_SupFig2.tiff1520KSupplemental Figure 2: Singlet gating based on cell length vs. DNA content (as measured by Ir intercalator). This gate excludes events with increased cell length relative to DNA content which are frequently cell doublet events.
CYTOA_22075_sm_SupFig3ABC.tiff1520KSupplemental Figure 3: Mass cytometry cell cycle assessment in stimulated murine splenic T cells and the murine lymphoma cell line A20. A) A plot of IdU vs. p-Rb(S807/811) allows for gating of G0 and G1 phase populations. (B) A plot of IdU incorporation vs. cyclin B1 allows gating of G1, S, and G2/M populations. (C) p-HH3(S28) defines an M-phase population. (D) Percentage of A-20 cells in each cell cycle phase as measured by mass and fluorescence (Hoechst-pyronin Y) cytometry. Six separate aliquots were taken from a single culture and individually treated with IdU and prepared for mass or fluorescence cytometry analysis. Standard deviation is shown by error bars. Gating methodology is identical to that shown in Figure 4.
CYTOA_22075_sm_SupFig3D.tiff1520KSupplemental Figure 3: Mass cytometry cell cycle assessment in stimulated murine splenic T cells and the murine lymphoma cell line A20. A) A plot of IdU vs. p-Rb(S807/811) allows for gating of G0 and G1 phase populations. (B) A plot of IdU incorporation vs. cyclin B1 allows gating of G1, S, and G2/M populations. (C) p-HH3(S28) defines an M-phase population. (D) Percentage of A-20 cells in each cell cycle phase as measured by mass and fluorescence (Hoechst-pyronin Y) cytometry. Six separate aliquots were taken from a single culture and individually treated with IdU and prepared for mass or fluorescence cytometry analysis. Standard deviation is shown by error bars. Gating methodology is identical to that shown in Figure 4.
CYTOA_22075_sm_SupFig4.tiff1520KSupplemental Figure 4: Comparison of gating strategies for mass cytometry and fluorescence cytometry. (A) NALM-6 cells were analyzed by mass cytometry with gating based on p-Rb vs. IdU, and cyclin B1 vs. IdU. (B) The same cells were analyzed in a fluorescence experiment utilizing gating based on the same markers (p-Rb(S807/811), Alexa647; cyclin B1, FITC; anti-BrdU/IdU, detected with anti mouse-IgG, PE-Cy7). (C) In the same fluorescence experiment, cells were also analyzed utilizing the traditional cell cycle markers Hoechst and pyronin Y. A comparison of p-Rb(S807/811) and pronin Y staining is also shown (right).
CYTOA_22075_sm_SupFig5.tiff1520KSupplemental Figure 5: Three distinct p-Rb(807/811) populations are present in NAML6 cells. Biaxial plots of p-Rb(S807/811) vs. each of the cell cycle markers tested in this report.
CYTOA_22075_sm_SupFig6.tiff1520KSupplemental Figure 6: Comparison of G2 phase cells across myeloid differentiation. Top left, “branch” of myeloid development from hematopoietic tree shown in Figure 6. Top center, population annotations and expression levels of population defining surface markers for the myeloid “branch” (node color and node sizing scaling as described in Figure 6). Top right, distribution of cells in each phase of the cell cycle in the myeloid “branch” of the tree. Node sizing as described in Figure 7A. Red box, biaxial plots derived from the mature myelocyte population from SPADE analysis. Cyclin B1 expression was compared to IdU, cyclinA, p-Rb(S807/811), and p-CDK1(Y15). Blue box, biaxial plots derived from the pro-myelocyte population from SPADE analysis. Cyclin B1 expression was compared to IdU, cyclinA, p-Rb(S807/811), and p-CDK1(Y15).
MIFlowCyt-Item-Location.doc1058KSupporting Information: MIFlowCyt

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