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. 2022 Apr 13;3(2):101309.
doi: 10.1016/j.xpro.2022.101309. eCollection 2022 Jun 17.

Quantification of unperturbed phosphoprotein levels in immune cell subsets with phosphoflow to assess immune signaling in autoimmune disease

Calvin Krollmann  1 , Kevin Cieslak  1 , Ruth-Miriam Koerber  1 , Hella Luksch  2 , Angela Rösen-Wolff  2 , Peter Brossart  1 , Lino L Teichmann  1
Affiliations

Quantification of unperturbed phosphoprotein levels in immune cell subsets with phosphoflow to assess immune signaling in autoimmune disease

Calvin Krollmann et al. STAR Protoc. .

Abstract

Activation of innate immune sensors by endogenous DNA and RNA can lead to autoimmune and autoinflammatory diseases. Quantification of the unperturbed phosphoprotein content in immune cells provides insight into the spontaneous activity of immune signaling pathways triggered by nucleic acid recognition. Here, we present a phosphoflow protocol for measuring phosphoproteins in mouse models of autoimmunity that incorporates strategies to preserve native phosphoprotein levels during sample collection and to reliably detect low signaling activity common in chronic disease states. For complete details on the use and execution of this protocol, please refer to Jütte et al. (2021).

Keywords: Flow Cytometry/Mass Cytometry; Immunology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Gating strategy to identify relevant immune cell subsets by flow cytometry Gating of cytotoxic (TCRβ+CD8α+) and helper (TCRβ+CD8α-) T cells, classical type 1 (CD11c+MHC-II+CD8α+CD11b-) and type 2 (CD11c+MHC-II+CD8α-CD11b+) dendritic cells after removal of duplicates.
Figure 2
Figure 2
Quantification of p-IRF-3 and p-Stat1 in immune cell subsets (A) Increased basal IRF-3 signaling activity in splenic cDCs from Trex1−/− mice, which develop chronic autoimmunity caused by the recognition of unmetabolized cytosolic DNA by cGAS, compared to those from wild type mice. Fold change of p-IRF-3 mean fluorescence intensity (MFI) of cDCs in instantly fixed spleens from wild type mice (n = 3) andTrex1−/− mice (n = 3). (B) Histograms display representative results for p-IRF-3 (Ser396) of gated cDCs in instantly fixed spleens. Each histogram shows an overlay of dephosphorylated (gray shaded) and native (colored) cells. (C and D) As in (A) and (B) but for p-Stat1 (Tyr701) in T cells. Data in bar graphs are represented as mean ± SEM. Individual fold change values were calculated as ΔMFIi = (MFIi, native – MFIi, dephosphorylated) divided by the average of ΔMFIi values obtained from wild type mice. Statistically significant differences were determined by two-tailed t-test (∗p < 0.05).
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References

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