Apc conjugated anti cd11c

FL-DCs were prepared as previously described (37 (link)), with some modifications. In brief, bone marrow cells were cultured at a concentration of 3×10⁵ cells/ml in RPMI-1640 medium containing 10% FBS supplemented with 200 ng/ml recombinant murine Flt3L (rmFlt3L, PeproTech, Rocky Hill, NJ, USA) and 2-mercaptoethanol (50 μM, Sigma-Aldrich), for 8 d. Cells were treated with MEHP, GW9662 (PPARγ antagonist; Tocris, Bristol, UK), or GW1929 (PPARγ agonist; Sigma-Aldrich) at various concentrations or with 0.1% ethanol (vehicle control) at the beginning of day 1 of culture. The medium containing rmFlt3L and/or chemicals or 0.1% ethanol was refreshed on days 4 and 6. On day 8, the cells were stained with the following antibodies, for phenotype analysis using flow cytometry: PE-conjugated anti-CD86 (GL-1; eBioscience), PerCP-cy5.5-conjugated anti-CD24 (M1/69; BioLegend), BV421-conjugated anti-CD45RA (14.8; BD Biosciences), APC-conjugated anti-CD11c (N418), APCcy7-conjugated anti-CD11b (M1/70; BioLegend), Alexa Fluor™ 700-conjugated anti-MHC II (M5/114.15.2), and LIVE/DEAD™ Fixable Red. For functional analysis, day-8 FL-DCs were washed and then stimulated with CpG1826 (10 μg/ml, InvivoGen, Carlsbad, CA, USA) for 24 h. The supernatants of FL-DCs were assessed for levels of cytokines using ELISA (R&D Systems).

BALF cells were incubated with 2.4 G2 mAb against FcγRII/III [23 (link)] and stained with APC-conjugated anti-CD11c (Biolegend, San Diego, CA, USA), BUV395-conjugated anti-CD11b (BD Biosciences, Franklin Lakes, NJ, USA), FITC-conjugated or PE-Cy7-conjugated anti-Ly6G (clone 1A8, BD Biosciences, Franklin Lakes, NJ, USA), FITC-conjugated anti-MHCII (I-A/I-E), PerCP-Cy5.5-conjugated or PE-conjugated anti-Ly6C(Biolegend, San Diego, CA, USA), and BV421-conjugated (Biolegend, San Diego, CA, USA) or PE-conjugated anti-Siglec-F (BD Biosciences, Franklin Lakes, NJ, USA) mAbs. The stained cells were analyzed on BD LSRII-green using BD FACSDiva software. Data analysis was completed using FlowJo software.

3M-052 and NHWD-870 were purchased from MedChemExpress Biological Technology Co., Ltd. (Shanghai, China). UiO-66 was purchased from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). RIPA lysis buffer and a BCA protein assay kit were acquired from Beyotime Biotechnology. A Cell Counting Kit-8 (CCK-8) cell counting kit and Annexin V-FITC/PI apoptosis detection kit were purchased from APE x BIO Technology (Houston, USA). Fetal bovine serum and RPMI-1640 were purchased from Procell Life Technology (Wuhan, China). The anti-BRD4(ab128874), anti-c-MYC(P01106), anti-PD-L1(Q9EP73), anti-cleaved caspase-3(P42574), anti-CRT(P27797), anti-β-actin(P60709), anti-HMGB1(GB11103-100), anti-TNF-α(GB11188-100), and anti-IL-6(GB11117-100) antibodies were purchased from Abcam (Cambridge, UK), Abmart (Shanghai, China), Cell Signaling Technology, Inc. (Danvers, MA, USA), Proteintech (Wuhan, China), and Servicebio Technology (Wuhan, China), respectively. Monoclonal antibodies, including PE-conjugated anti-CD86, APC-conjugated anti-CD80, APC-conjugated anti-CD11c, FITC-conjugated anti-CD8, PE-conjugated anti-CD4, and Alexa 647-conjugated anti-Foxp3 antibodies, were purchased from Biolegend (California, USA) and Servicebio Technology (Wuhan, China).

An anti-FcR antibody was used to block the non-specific staining. Cells were subsequently immunostained with APC-conjugated anti-CD11c, APC-cy7-conjugated anti-CD86, and FITC-conjugated anti-MHC-II antibodies (Biolegend, USA).