Flow Cytometry Analysis of Immune Cells

Spleen, mesenteric lymph node (mLN), and ankle joints were processed for flow cytometry as we previously described [13 (link)]. The following antibodies were used for surface molecular staining: BV510-anti-CD45 (30F11), FITC-CD4 (JK1.5), FITC-Lineage (CD3/GR-1/CD11b/CD45R/TER-119), BV421-CD127 (A7R34), PerCP/Cy5.5-KLRG1 (2F1), PE-ST2 (RMST2-2), APC-ICOS (C398.4A), APC-NK1.1 (PK136), PerCP/Cy5.5-Ly6G(1A8), FITC-CD11b(M1/70), APC-F4/80 (BM8, all from BioLegend), and PE-SiglecF (1RNM44N, eBioscience). For intracellular staining, cells were fixed and permeabilized by the FoxP3/Transcription Factor Staining Buffer (eBioscience) and then stained with PE/Cy7-anti-T-bet (4B10), PE-anti-RORγt (AFKGS-9), PerCP/Cy5.5-IFN-γ (XMG1.2, BD Biosciences), APC-IL-4 (11B11, BioLegend), and AF647-IL-17A (TC11-18H10, BD Biosciences) at 4°C for 30 min. In some experiments, mLN cells and splenocytes were plated on a 12-well plate and incubated with cell activation cocktail (with Brefeldin A, BioLegend) at 37°C for 4 h before staining.

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Zhang Y., Qin Y, & Chen Z. (2021). Neuromedin U Suppresses Collagen-Induced Arthritis through ILC2-Th2 Activation. Journal of Immunology Research, 2021, 5599439.

Publication 2021

FITC-CD4 FITC-Lineage FoxP3/Transcription Factor Staining Buffer PE-anti-RORγt APC-IL-4 cell activation cocktail (with Brefeldin A,

Af647 Ankle joints Anti t Antibodies Brefeldin a Buffer Cd11b Cell Cy5 5 Fitc Flow cytometry Icos Ifn γ Il 17a Intracellular Klrg1 Lymph node Mesenteric Rorγt Spleen Transcription factor

Corresponding Organization : University of Science and Technology of China

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Variable analysis

independent variables

Tissue type (spleen, mesenteric lymph node, ankle joints)

dependent variables

Immune cell populations and their phenotypes as measured by flow cytometry

control variables

Antibodies used for surface and intracellular staining
Cell fixation and permeabilization protocol
Cell activation and incubation conditions

controls

Positive control: Cell activation cocktail (with Brefeldin A) used to stimulate cells before staining

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