ACK lysed splenocytes from three Zbtb46GFP/WT mice were isolated and enriched for CD19− CD3− cells. Cells were stained for 30 min at 4°C with the following 9 backbone antibodies and then washed: 7-AAD, SiglecH-AF647 (clone 551), CD11c-APC/Cy7 (clone N418), Bst2-PacBlue (clone 927), MHC-II-BV510 (clone M5/114.15.2), CD11b-BV785 (clone M1/70), XCR1-BV650 (clone ZET), CD19-Bio (clone 6D5), CD3-Bio (clone 17A2). The backbone labeled cells were then stained with 255 different PE-conjugated antibodies using the LegendScreen Mouse PE kit (BioLegend) following the manufacturer’s instructions. We used the Infinity Flow pipeline to combine overlapping flow cytometry panels and simultaneous analyze the co-expression patterns of hundreds of surface-expressed proteins across millions of individual cells as described in Becht et al. 54 (link). Clustering was performed using the Flowsom clustering method, with the parameters FlowSOM metacluster k = 7.
Protocol detail
Profiling Zbtb46+ Immune Cell Subsets
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Corresponding Organization : SIB Swiss Institute of Bioinformatics
Other organizations : National Institute of Allergy and Infectious Diseases
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Variable analysis
independent variables
- Zbtb46 genotype (Zbtb46^GFP/WT)
- Expression of surface-expressed proteins on CD19^- CD3^- cells
- Incubation time (30 min)
- Temperature (4°C)
- Staining with 9 backbone antibodies
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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