Cd44 apc im7

Lymphocytes were isolated from the C57BL/6 mouse spleens and MLNs in single-cell suspensions. The cells were stained with fluorescence-labeled antibodies against CD4-FITC and CD44-APC at 4°C for 30 min. The samples were stained with antibodies against Foxp3-PE (NRRF30, eBioscience Inc., San Diego, CA, USA) at room temperature for 30 min in the dark after using a Foxp3 fixation/permeabilization kit (BD Biosciences, San Diego, CA, USA) [29 (link)]. The lymphocytes collected from the Foxp3^GFP mouse spleens were stained with fluorescence-labeled antibodies against CD4-PE and CD62L-APC at 4°C for 30 min. Flow cytometry was performed on an LSRII cytometer (BD Biosciences) and analyzed with the FlowJo 7.6 software. Anti-mouse CD4-FITC (GK1.5) and Foxp3-PE (NRRF30) were purchased from eBioscience. CD62L-APC (MEL-14) was purchased from BD Biosciences. CD4-PE (GK1.5) and CD44-APC (IM7) were obtained from BioLegend Inc. (San Diego, CA, USA).

Samples were first stained with Fixable Viability Stain 440 UV (BD Biosciences), and incubated for 15 min on ice with anti‐CD16/32 antibody (clone 2.4G2) to block Fc receptors. Samples were then stained with the following antibodies CD3ε‐BB700 (145‐2C11), CD4‐BUV737 (RM4‐5), CD8α‐BV650 (53‐6.7), CD25‐BUV395 (PC61), CD27‐PE (LG.3A10), CD69‐BV605 (H1.2F3), CD117‐PE‐CF594 (2B8), and CD161‐APC‐Cy7 (PK136), all from BD Biosciences, with CD5‐PE‐Cy7 (53‐7.3), CD24‐A700 (M1/69), CD44‐APC (IM7), and CD71‐BV421 (RI7217), all from Biolegend, and with TCRδ‐PE‐Cy5 (GL‐3) from Invitrogen. Multiparameter FACS acquisition was performed on a Fortessa LSRII SORP system (BD Biosciences) and data analyzed with FACSDiva 9.0 (BD Biosciences) and FlowJo 10.7.1 (BD Biosciences) software. Doublets were systematically excluded based on side scatter (SSC) and forward scatter (FSC) parameters.

Hybridoma cell line producing H35 was grown in IMDM culture medium supplemented with 2% of IgG-depleted FCS, 10 mM HEPES, and 50 μM β-mercaptoethanol at 37°C in 5% CO2 atmosphere. The anti-CD8β mAb was purified from cell culture supernatant of the H35 hybridoma on Protein G sepharose column (Amersham). Before immuno-staining, dead cells were excluded with the fixable viability dye eFluor506 (eBioscience). All antibodies used for flow cytometry experiments were diluted in an anti-mouse Fc receptor mAb purified from the supernatant of culture of the 2.4G2 hybridoma. Fluorochrome-conjugated monoclonal antibodies for flow cytometry were purchased from eBioscience: CD3-eFluor450 (eBio500A2), CD11c-PECy7 (N418), CD8α PerCpCy5.5 (53-6.7), CD62L Alexa Fluor700 (MEL-14), CD44 APC (IM7), and IFNγ PECy7 (XMG1.2) or BioLegend: CD19 PE (6D5) and CD4 APCCy7 (RM4-5). Analyses were performed with the FACSLSRII or FACSCanto machines (Becton Dickinson) using the FACSDiva software for the acquisition and the FlowJo software for data analyses.