Anti cd62l apc

The following fluorochrome-labeled mouse antibodies were used according to the manufacturers’ protocols: PB or Percp/cy5.5 anti -CD4, PB or PreCP/cy5.5 anti- CD8, PE or PE/cy7 anti- CD3, APC anti-CD62L, Fitc or PE/cy7 anti- CD44 (Biolegend). Cells were sorted on a SORP-FACS-AriaII and analyzed using FACSDiva (BD Biosciences) and FlowJo (Tree Star) software. Sorted cells were centrifuged (450g for 10 minutes) before RNA extraction.

The flow cytometry was used to analyze cell populations by incubating cells with different antibodies or fluorescence. The single cell suspensions were incubated with anti-CD16/32 (Biolegend, San Diego, CA, USA) prior to staining to block non-specific binding to the Fc receptor. Cells were incubated for 30 min at 4 ℃ for surface staining. For intracellular cytokine staining, cells were incubated with a stimulating/blocking cocktail for 4 h prior to surface staining and cytokine staining was performed after treatment with fixation/permeabilization kits.
The following antibodies were used: FITC anti-CD45, APC-Cy7 anti-CD11b, Brilliant Violet 421 anti-Ly6C, PE anti-CD44, PE anti-F4/80, APC anti-Ly6G, APC anti-CD62L, APC anti-CD25, PE anti-IL-17A, and Zombie Aqua Fixable Viability Kit (Biolegend, San Diego, CA, USA).
For CFSE staining, cells were incubated with 1.25 μM CFSE (Biolegend, San Diego, CA, USA) for 10 min prior to incubation. For mitochondrial reactive oxygen species (mROS) assay, cultured cells were incubated with 5 μM MitoSOX (Invitrogen, Carlsbad, USA) for 30 min. For mitochondrial membrane potential (MMP) assay, cultured cells were incubated with JC-1 (1:200, Biyuntian, Wuhan, China) in buffer for 10 min. For mitochondrial mass assay, cultured cells were incubated with 5 μM mitoTracker (Invitrogen, Carlsbad, USA) for 30 min. Fluorescence was measured by flow cytometry.

Tetraethyl orthosilicate (TEOS), ethanol, ammonia, potassium permanganate (KMnO₄), and sodium carbonate (Na₂CO₃) were purchased from Macklin (China). Ovalbumin (OVA) was obtained from Sigma (USA). OVA-Cy5.5 was obtained from QIYUE BIOLOGY(China). DNA single strands A₅₀ and T₂₀ were brought from Tsingke Biotech (China). Enhanced CCK-8 kit, Lyso-Tracker green fluorescent dye, 4’,6-diamidino-2-phenylindole (DAPI) and BCA protein assay kit were purchased from Beyotime (China). Roswell Park memorial institute (RPMI) 1640 culture medium, dulbecco’s modified eagle medium (DMEM), and fetal bovine serum (FBS) brought from Procell (China). Anti-CD11c-APC, anti-MHC II-PE, anti-MHC I-PE, anti-CD80-Cy5.5, anti-CD86-FITC, FITC-anti-CD4, Cy5.5-anti-CD8a, PE-anti-CD44, APC-anti-CD62L and APC-anti-CD3 antibodies were supplied by BioLegend (USA). Enzyme-linked immunosorbent assay (ELISA) Kits for INF-γ, TNF-α, IL-4, and IL-6 were also purchased from BioLegend (USA). In addition, all animal experiments were determined eligible for the study and were approved by the Ethics Committee of Jinan University.