Ic5868p

IFN-γ (315-05), IL-4 (214-14), M-CSF (315-02) and GM-CSF (315-03) were purchased from Peprotech (Rocky Hill, NJ). LPS (from Escherichia coli, serotype 055:B5, L4005) was purchased from Sigma (St. Louis, MO). The primary antibodies and dilutions used were: Flag (Sigma, F3165, 1:2000), α-tubulin (Sigma, T6199, 1:2000) and β-actin (Sigma, A2228, 1:5000), β-TrCP(Cell Signaling Technology, 4394, 1:1000), Nesprin1 (Santa Cruz, sc-99065, 1:500), Nesprin2 (Santa Cruz, sc-365097, 1:500), LaminA/C (Santa Cruz, sc-7292, 1:1000), ubiquitin (Santa Cruz, sc-8017, 1:1000), CD11b (eBioscience, 17-0112, 1:100), CD86 (eBioscience, 11-0862, 1:100), CD45 (eBioscience, 48-0451, 1:100), F4/80 (eBioscience, 25-4801, 1:100), CD206 (R&D systems, FAB2535P, 1:100) and Arg1 (R&D systems, IC5868P, 1:100) were obtained from. human SUN1 (Abcam, ab103021, 1:1000), mouse SUN1 (Abcam, ab124770, 1:1000) and mouse SUN2 (Abcam, ab124916, 1:1000), and mouse SUN2 (Abcam, ab198981, 1:100, FCS). An antibody specific for human SUN2 (1:1000) was produced by Shanghai Immune Biotech (Shanghai, China).

After isolation, cells were stained with fixable live/dead dye (Life Technologies), blocked with anti-CD16/32, and stained with antibodies for CD45 (30-F11), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), CD11c (N418), Ly6C (HK1.4), F4/80 (BM8), CD64 (X54-5/7.1), XCR1 (ZET), SIRPα (P84), and/or major histocompatibility complex class II (MHCII, M5/114.15.2).
For intracellular iNOS and ARG1 production by myeloid cells, the cells were stained for extracellular markers and then fixed and permeabilized with 2% formaldehyde for 30 min. Intracellular staining for iNOS (CXNFT) and ARG1 (IC5868P, R&D SYSTEMS) was performed in the permeabilization buffer.
For intracellular cytokine production by T cells, the cells were stimulated for 6 h with phorbol myristate acetate and ionomycin in the presence of brefeldin A and monensin for the final 4 h. The cells were stained for extracellular markers and then fixed with fixation/permeabilization buffer, and intracellular staining for Foxp3 and IFN-γ was performed in permeabilization buffer. Fluorescence minus one (FMO) controls were used for all experiments. Data were collected on either an LSRII cytometer (BD) or an LSRFortessa (BD) and were analyzed using FlowJo (TreeStar).

After treatment with IFN-γ and 5-HMF, BV2 cells were harvested for flow cytometry detection as previously described
[35] (link). Briefly, cells were fixed, permeabilized, and incubated with rabbit anti-mouse iNOS (ab283668; Abcam) or sheep anti-mouse Arg-1 (10 μL, IC5868P; R&D Systems, Minneapolis, USA) at 4°C for 30 min. After being washed with the specified washing buffer, cells were harvested and detected with a FACS Caliber instrument (BD Biosciences, San Jose, USA), and the relative mean fluorescence in each group was analysed using Flow Jo software (version 10.0; Tree Star, Ashland, USA).