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1.
Fig. 4.

Fig. 4. From: Profiling antibody responses by multiparametric analysis of primary B cells.

Affinity heatmap of anti-H-2Kb hybridomas generated by k-means (n = 3) clustering (3,711 cells). The median profiles for each cluster are superimposed (black).

Craig M. Story, et al. Proc Natl Acad Sci U S A. 2008 November 18;105(46):17902-17907.
2.
Fig. 6.

Fig. 6. From: Profiling antibody responses by multiparametric analysis of primary B cells.

Affinity heatmap of antigen-specific cells identified in a mouse after two boosters indicates the diversity in antibody response. Both the binding curves and the isotypes were included in the hierarchical clustering (Euclidean/average) but separated in the figure for graphical clarity. The colored branches in the dendrograms indicate the cells that are most closely related. The KDapps (nM) calculated from a Langmuir curve fit for each clone are listed to the right of the data for each cell; these numerical values were not considered in clustering. OVA, ovalbumin.

Craig M. Story, et al. Proc Natl Acad Sci U S A. 2008 November 18;105(46):17902-17907.
3.
Fig. 3.

Fig. 3. From: Profiling antibody responses by multiparametric analysis of primary B cells.

Method for constructing an affinity heatmap. (A) Three examples of measured binding curves from the anti-H-2Kb clones are shown: (i) antigen-specific antibodies that exhibit saturation of binding (filled triangles), (ii) nonspecific/low-affinity antibodies (open circles), and (iii) antigen-specific antibodies that do not exhibit saturation of binding (filled squares). The composite fluorescent images correspond to each data point in the median-centered curves. The colored bands shown on the plots indicate the colors used in the corresponding density plots in B. (B) Construction of an affinity heatmap for the three clones in A. Each row represents the binding curve measured for the antibody secreted by one cell as a density plot. Antigen-specific antibodies that do not exhibit saturation show only blue coloring. Nonspecific or low-affinity antibodies have no coloring.

Craig M. Story, et al. Proc Natl Acad Sci U S A. 2008 November 18;105(46):17902-17907.
4.
Fig. 2.

Fig. 2. From: Profiling antibody responses by multiparametric analysis of primary B cells.

Binding curves distinguish similar hybridomas. (A) Representative composite fluorescent micrographs of one microarray and the corresponding cells in microwells (Y3, aqua; c127, magenta; c136, purple). The microarray was imaged after staining with anti-mouse IgG (red) and tetrameric MHC class I (H-2Kb) (green). The dashed white boxes indicate the physical boundaries of each microwell. (Scale bar: 50 μm.) (B) Graph of the antigen-to-antibody ratios measured for each element of the microarray shown in A as a function of the concentration of tetrameric H-2Kb across all seven replicates. The colors of the traces link them to the identity of the clones from which they are derived.

Craig M. Story, et al. Proc Natl Acad Sci U S A. 2008 November 18;105(46):17902-17907.
5.
Fig. 1.

Fig. 1. From: Profiling antibody responses by multiparametric analysis of primary B cells.

Measurements of the apparent affinities of antibodies produced by single cells. (A) Representative composite micrographs from five antibody microarrays produced by microengraving and the corresponding region of wells containing antiovalbumin hybridomas stained with carboxyfluorescein succinimidyl ester. The microarrays are labeled with anti-mouse Ig (red) and ovalbumin (OVA; green). The dashed white boxes indicate the physical boundaries of each microwell. (Scale bar: 50 μm.) (B) Box plot of the antigen-to-antibody ratios measured for 3,461 cells as a function of the concentration of antigen applied to five replicate microarrays. (Inset) Graph of the median binding curves measured by microengraving (dashed line) and by ELISA (solid line) for the purified antibody.

Craig M. Story, et al. Proc Natl Acad Sci U S A. 2008 November 18;105(46):17902-17907.
6.
Fig. 5.

Fig. 5. From: Profiling antibody responses by multiparametric analysis of primary B cells.

Profiles of antibody responses generated by immunized mice. (A) Immunization schedule used for the three mice profiled. (B) Graphical profiles of the populations of single cells characterized from each mouse. The total area of each circle is proportional to the number of cells enumerated with the phenotypes indicated. Single cells expressing either B220 or IgM on their surfaces were classified as B cells. The innermost light blue-green circle represents the subset of individual secreting B cells for which a complete binding curve was obtained. The distribution of isotypes for this subset is indicated to the left of the light blue-green circles for each mouse. Red circles represent the subset of individual antibody-secreting B cells classified as antigen-specific (with a KDapp < 100 nM). The distribution of isotypes in each of these subsets is shown to the right of the red circles for each mouse. The quality of the microengraved microarrays can be estimated by comparing the number of single secreting B cells that yielded a complete set of affinity data with the total number of single secreting B cells detected (i.e., relative size of the light blue-green circle with respect to the enclosing isotype+ circle); here (from left to right), 68.5%, 39%, and 20.7%.

Craig M. Story, et al. Proc Natl Acad Sci U S A. 2008 November 18;105(46):17902-17907.

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