Single-cell suspensions were stained using mouse-specific Abs, including FITC–anti-CD11b (BD Biosciences), Alexa 647–anti-IL-23p19 (eBioscience), PE–anti-TNF-α, PE/Cy7–anti-Ly6C for macrophages and subsets. For CD4 T cell and subset staining, cells were stained with FITC–anti-CD4, PE/Cy7–anti-Thy1.2, APC–anti-IFN-γ, and PE–anti-IL-17. Intracellular and intranuclear staining was performed as described previously (15 (link)). For macrophages treated with 2-D-gal in vitro, cells were stained with Bio-ICAM followed by streptavidin eFluor 450 (eBioscience) and APC–anti-CD11b, FITC–anti-CD80, and PE–anti-CD86. 2D2 CD4 T cells were identified by staining with APC–anti-CD4, PE/Cy7–Vβ11. DQ-Ovalbumin (DQ-OVA, Invitrogen) was detected in FITC channel. For macrophages loaded with Eα-GFP peptides, cells were stained with bio-YAe (eBioscience), followed by streptavidin eFluor 450 (eBioscience) and APC–anti-CD11b (Biolegend).
To determine the degree of ERK1/2 intracellular signaling, we performed phospho-flow studies according to the protocol from BD Biosciences. Intracellular pERK1/2 was stained with Rabbit anti-ERK1 (T202/Y204)/ERK2 (T185/Y187) (R&D systems), followed by Alexa488–goat anti-rabbit IgG (Invitrogen).
Data were acquired on a BD LSRII flow cytometer and analyzed using FlowJo software (Tree Star, Inc.).
To determine the degree of ERK1/2 intracellular signaling, we performed phospho-flow studies according to the protocol from BD Biosciences. Intracellular pERK1/2 was stained with Rabbit anti-ERK1 (T202/Y204)/ERK2 (T185/Y187) (R&D systems), followed by Alexa488–goat anti-rabbit IgG (Invitrogen).
Data were acquired on a BD LSRII flow cytometer and analyzed using FlowJo software (Tree Star, Inc.).