Anti cd62l fitc

20 μl of peripheral blood were collected from the tail vein and collected in FACS buffer. Splenocytes were prepared by grinding spleens between frosted-end microscopic slides in petri dishes containing RPMI medium supplemented with 5% FCS. For analysis of pulmonary cells, mouse lungs were perfused through the pulmonary artery with 5 ml PBS. The lungs were excised, cut into small pieces and incubated for 60 min at 37°C in RPMI medium supplemented with 5% FCS and 200 μg/ml Collagenase D (Roche Diagnostics, Risch, Switzerland) and 10 μg/ml DNAse I (Sigma, Deisenhofen, Germany). The suspension was resuspended with a Pasteur pipette every 15 min. The reaction was stopped by 5 min incubation at 37°C with 5 mM EDTA. Lung cells were then mechanically separated in 100 μm mesh sized cell strainers.
Erythrocytes were lysed with Tris-buffered 0.15 M ammonium chloride for 1 min (lung) or 7 min (blood, spleen). Unspecific binding sites were blocked with anti-FcγR at 4°C for 10 min. The cells were extracellularly stained with anti-CD4-FITC, anti-CD4-APC, anti-B220-APC, anti-CD3-PE, anti-CD8-PerCP-Cy5.5, or anti-CD62L-FITC (all from BD, Heidelberg, Germany), respectively, for 1 h at 4°C. Cells were analyzed on an Accuri C6 cytometer or fixed in 1% PFA for 30 min and analyzed on an LSR-II cytometer (both BD). Absolute cell numbers of blood cell populations were measured with the C6 cytometer.

NOD mice were purchased from Beijing HFK Bioscience CO., LTD (Beijing, China). All mice were housed in a specific pathogen-free environment and all animal experiments were approved by the Institutional Animal Care and Use Committee. Traditional Chinese medicine formula, Ginseng and Astragalus granule (GAG), composed of Ginsenosides of it's stem and leaves, Schisandra, Astragalus, Yam, Radix rehmanniae, Ophiopogon japonicus, Poria, Radix trichosanthis, Rhizoma alismatis and Chinese wolfberry, was purchased from Lunan Houpu Pharmaceutical Co. Ltd (Lunan, China). Cyclosporin A (CsA) was purchased from the Guangdong Provincial Hospital of Traditional Chinese Medicine (Guangzhou, China). Anti-CD4-PE, anti-Foxp3-APC, anti-CD8-PE, anti-CD44-PE-Cy5, anti-CD62L-FITC, anti-CD8-FITC, anti-CD122-PE, and anti-PD-1-APC mAbs were purchased from BD Biosciences (BD Biosciences, San Jose, CA) or eBioscience (eBioscience, San Diego, CA).

CIK cells were resuspended at 2 × 10⁵ cells per 100 μL of phosphate-buffered saline (PBS) and incubated for 30 min at 4 °C with the following anti-human antibodies: anti-CD3-PE-Cy5, anti-CD4-FITC, anti-CD8-PE-CF594, anti-CD25-APC, anti-CD56-PE-Cy7, anti-CD45RO-APC, and anti-CD62L-FITC (all from BD Bioscicence). The cells were analyzed using a CytomicsTM FC500 Flow Cytometer (Beckman Coulter, USA). Data analysis was performed with CXP analysis software (Beckman Coulter, USA).