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. 2024 Feb 8;16(3):641–663. doi: 10.1038/s44321-024-00028-y

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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Figure EV2 — (A) Joint-footpad cells from non-infected and CHIKV-infected animals were harvested at 6 dpi and were subjected to stimulation with ionomycin and Phorbol Myristate Acetate (PMA) for 4 h. Stimulated cells were subsequently stained for the presence of GM-CSF and IFNγ in CD4⁺CD44⁺ memory/effector T cells. Representative electronic gating strategy to isolate CD4⁺CD44⁺ T cells from joint-footpad cells. Plots shown are concatenated from CHIKV-infected samples. (B) Representative flow cytometry plot illustrating the gating strategy in the identification of GM-CSF and IFNγ within the CD4⁺CD44⁺ T cells. (C) Dotplots showing the numbers of the various identified subsets within the joint-footpads. Data were from biological replicates obtained from two independent experiments. All data are presented as mean ± SD. Data comparisons between the groups were performed with non-parametric Mann–Whitney U test (two-tailed). IFNγ⁺ T cells, ***P = 0.0000000499; GM-CSF⁺ T cells, ***P = 0.0000000499; IFNγ⁺GM-CSF⁺ T cells, ***P = 0.0000000499; IFNγ^-GM-CSF⁺ T cells, ***P = 0.0000000499. (D) Fresh blood was obtained from non-infected animals. Red blood cells were subsequently lysed and live cells were stained with a commercially available live/dead dye before being stained with a cocktail of antibodies targeting surface markers CD45, CD11b, Ly6G, Ly6C, CD64, and MHCII. Live CD45⁺ cells were firstly identified, and non-neutrophils were gated next. CD11b⁺Ly6C⁺ monocytes were identified from the non-neutrophils and were shown to be low in CD64 and MHCII expression (Q4). Plots shown are representative of a single non-infected mouse. (E) Peripheral whole blood was obtained from non-infected animals and were subjected to GM-CSF and IFNγ stimulation after removal of the red blood cells. Stimulated cells were harvested 24 h later and stained for the identification of monocytes and macrophage subsets. Representative flow cytometry plots showing the gating strategy used to identify CD64⁺MHCII⁺ cells among the precursor CD11b⁺Ly6C⁺ monocytes.