Pe anti mouse cd62l

After six months of i.p. injection of the pristane, mice were sacrificed and the splenocytes were isolated from the spleens by density gradient centrifugation using LymphoprepT^M reagent (Axis-shield, Oslo, Norway). Cells were stained by fluorescence-conjugated antibodies (Abs) including APC-anti-mouse CD3, FITC-anti-mouse CD4, APC-anti-mouse OX40, PE-anti-mouse ICOS, AF700-anti-mouse CD38 (from eBioscience, Waltham, MA, USA), FITC-anti-mouse CD44, PE-anti-mouse CD62L, APC-anti-mouse ICAM-1, and FITC-anti-mouse IgG1 (from BD Biosciences). Cells were incubated at 4°C for 30 min and acquired on an LSR Fortessa (BD Biosciences). For intracellular staining, cells were fixed and permeabilized using the Transcription Factor Buffer Set (BD, Biosciences) according to the manufacture's protocol. Cells were incubated with PE-Cy7-anti-mouse Foxp3 for 40 min. Cells were acquired using an LSRFortessa flow cytometer (BD, Biosciences). Data analysis was carried out with FlowJo 7.6 software (Tree Star, Ashland, OR, USA).

For flow cytometry, fresh blood was collected in anticoagulant tubes (BD, 8215736) and peripheral blood mononuclear cells (PBMC) were obtained by lymphocyte isolate medium (KeyGEN BioTECH, kga831). Cells were washed and treated with the Fixation/Permeabilization Solution (BD) for Ki67 detection, incubated with flow cytometry antibodies (BD Pharmingen), and resuspended in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA). Then cells were determined with BD FACS Calibur Flow Cytometer. Antibody collocations were as follows:
(i)

CD4⁺ CD25⁺ T cells: FITC Anti-Mouse CD4 (eBioscience, clone: RM4–4), APC Anti-Mouse CD25 (eBioscience, clone: PC61.5);

(ii)

CD4⁺ CD44⁺ T cells: FITC Anti-Mouse CD4 (eBioscience, clone: RM4–4), PerCP Anti-Mouse CD44 (BioLegend, clone: IM7);

(iii)

CD4⁺ CD62L⁺ T cells: FITC Anti-Mouse CD4 (eBioscience, clone: RM4–4); PE Anti-Mouse CD62L (BD, clone: MEL-14);

(iv)

CD4⁺ Ki67⁺ T cells: PE Anti-Mouse CD4 (BD, clone: RM4–5); FITC Anti-Mouse Ki67 (BioLegend, clone: 16A8).