Cell size assays for mass cytometry

Abstract

Mass cytometry offers the advantage of allowing the simultaneous measurement of a greater number parameters than conventional flow cytometry. However, to date, mass cytometry has lacked a reliable alternative to the light scatter properties that are commonly used as a cell size metric in flow cytometry (forward scatter intensity-FSC). Here, we report the development of two plasma membrane staining assays to evaluate mammalian cell size in mass cytometry experiments. One is based on wheat germ agglutinin (WGA) staining and the other on Osmium tetroxide (OsO₄ ) staining, both of which have preferential affinity for cell membranes. We first perform imaging and flow cytometry experiments to establish a relationship between WGA staining intensity and traditional measures of cell size. We then incorporate WGA staining in mass cytometry analysis of human whole blood and show that WGA staining intensity has reproducible patterns within and across immune cell subsets that have distinct cell sizes. Lastly, we stain PBMCs or dissociated lung tissue with both WGA and OsO₄ ; mass cytometry analysis demonstrates that the two staining intensities correlate well with one another. We conclude that both WGA and OsO₄ may be used to acquire cell size-related parameters in mass cytometry experiments, and expect these stains to be broadly useful in expanding the range of parameters that can be measured in mass cytometry experiments. © 2016 International Society for Advancement of Cytometry.

Keywords: CyTOF; HEK293; U87; WGA; cell size; flow cytometry; mass cytometry; whole blood.

Figure 1

Establishing Wheat Germ Agglutinin Staining Intensity as a Metric for Cell Size. A-C: Imaging of A488-WGA-stained HEK293 cells. Representative fields of view are shown for 20X fluorescence images (intensity adjusted image displayed for illustration purposes only) (A) or processed images (B). Individual cells were identified from the processed images and the integrated fluorescence intensity and area were calculated. Data for each cell are shown in (C). Pseudo volume is cell area raised to the 3/2 power. D, E: WGA-stained HEK293 (D) and U87 cells (E) were subjected to flow cytometry (see Methods), and the correlation between FSC and WGA staining intensity is shown. F–H: Blood samples were subjected to flow cytometry analysis and gating to discriminate between granulocytes, monocytes and lymphocytes was applied based on FSC vs. SSC data (F) or WGA vs. SSC data (G). Consistency of cell type proportions between the two gating strategies are shown in (H). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

WGA intensity in mass cytometry analysis of human whole blood. Whole blood was stained with a panel of antibodies to identify major immune subsets together with WGA-A488 detected with an Ho165-tagged anti-A488 secondary antibody, and the data were visualized using SPADE. WGA staining in a representative sample is shown across the SPADE tree as fold change relative to MMO control (A). Major immune populations are delineated on the tree based on canonical marker expression. The median normalized WGA staining intensity is shown for the identified immune cell subsets in 8 separate individuals (B), and the average intensity is correlated with the published diameters of the corresponding cell subsets (C and Table 2). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Validation of OsO₄ in Mass Cytometry (A) Fixed PBMCs were stained with increasing concentrations of OsO₄ and median signal intensity for all gated cell singlets was evaluated for all osmium isotopic mass channels and compared to natural isotopic abundance. B-E: Resting and PMA-stimulated PBMCs were fixed and stained with a panel of antibodies against surface markers and intracellularly with an antibody stained against phospho-p38. OsO₄ staining was performed either before or after antibody staining (B). The data were visualized using viSNE to map the multi-dimensional data to two-dimensional space (C-D). Os192 expression is shown across all populations on the map (C) and major immune populations on the map were manually identified using global gates based on canonical marker expression patterns (D). Phospho-p38 expression is shown for the gated T cell population (E), and the overlaid histograms are colored based on the fold change between the stimulated and non-stimulated control samples. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Using WGA and OsO₄ in Mass Cytometry. A-D: Whole blood was stained with a panel of antibodies to identify major immune subsets and co-stained with WGA-A488-Ho165 and OsO₄, and the data were analyzed using SPADE. Ho165-WGA (A) and Os192 (B) staining intensity are shown across the SPADE tree for a representative sample, with major immune populations delineated based on canonical marker expression. (C) Immune population from a representative sample overlaid on biaxial plot dot plot scaled to show comparative expression of WGA against Os192. (D) Correlation of normalized median WGA-Ho165 and Os192 signal intensity across immune cell subsets in 3 individuals. E,F: A dissociated human lung sample was stained with a panel of antibodies to identify major cell subsets and co-stained with WGA-A488-Ho165 and OsO₄, and the data were analyzed using SPADE. Ho165-WGA (E) and Os192 (F) staining intensity are shown across the SPADE tree with major populations delineated based on canonical marker expression. [Color figure can be viewed at wileyonlinelibrary.com]