Pe conjugated anti cd4

Mouse spleens were removed under aseptic conditions, cut into pieces and filtered with a 200-mesh nylon membrane to generate single cells. Lymphocytes were separated by gradient centrifugation using a mouse lymphocyte separation kit (Tbdscience, Beijing, China), washed with flow staining buffer (Thermo Fisher Scientific, Waltham, MA, USA) and counted. A total of 1 × 10⁶ lymphocytes were re-suspended in 50 μL of flow staining buffer, and 1 μL each of FITC-conjugated anti-CD3 (BioLegend, San Diego, CA, USA), PE-conjugated anti-CD4 (BioLegend), and APC-conjugated anti-CD8 (BioLegend) antibodies was added and incubated for 30 min at room temperature in the dark. Cells were washed twice with flow staining buffer and re-suspended in 400 μL of buffer, and samples were analysed using a NovoCyte flow cytometer (ACEA NovoCyte, San Diego, CA, USA).

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).

Th17 (CD4, IL-17), Treg (CD4, CD25, Foxp3), SMILE, AMPK, mTOR, pSTAT3 705, pSTAT3 727 and germinal center markers (CD3, B220, PNA) expression levels were analyzed by confocal microscopy. Tissue Sects. (7 μm thick) were fixed in methanol-acetone and stained with phycoerythrin (PE)-conjugated anti-CD4 (Biolegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti CD4 (BD Bioscience, San Diego, CA, USA), PE-conjugated anti-interleukin-17 (eBioscience, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-CD25 (Biolegend), PE-conjugated anti-Foxp3 (Thermo Fisher Scientific, Waltham, MA, USA), PE-conjugated anti-phosphorylated Stat3 (Tyr 705, Ser 727, BD Bioscience), APC-conjugated anti-B220 (eBioscience), Alexa 594-conjugated anti-PNA (Thermo Fisher Scientific), SMILE (Abcam, Cambridge, UK), AMPK (Abcam) and mTOR (Cell Signaling). After overnight incubation at 4℃, the stained sections were analyzed on a Zeiss confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany) at 200× magnification and Zen Blue edition software.