Fitc conjugated cd3

Colon tissue was collected from acute colitis mice and cut into pieces in 1 mg/ml of collagenase IV; collagenase digestion was performed in collagenase solution for 45 minutes at 37 °C. The eluted cells were then passed through a 70-μl cell strainer and collected for 10 minutes at 1400 rpm at room. The cells were incubated in 10% donkey serum and Fc blocked (BD Pharmingen, San Jose, CA) for 20 minutes at 4 °C and then stained with fluorescent-conjugated antibodies. FITC-conjugated CD3, APC-conjugated CD25, PE-conjugated CD4 (OX-38), and APC-conjugated Foxp3 were purchased from BD Pharmingen (San Diego, CA) and used for the FACS analysis. The cells were analyzed with the FACSCaliber cytometer (BD Bioscience, San Diego, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).

The anti-human monoclonal antibodies (mAbs) for flow cytometry were fluorescein iso-thiocyanate (FITC)-conjugated CD3, phycoerythrin-cyanine 5-conjugated CD56, phycoerythrin (PE)-conjugated CD11a, PE-conjugated CD16, PE-conjugated CD107a, PE-conjugated CD279, PE-conjugated CD335, PE-conjugated CD337, PE-conjugated CD314 and IgG1 isotype control, purchased from BD Biosciences (San Jose, CA, USA), and anti-human Abs PE-conjugated CD159c, PE-conjugated CD159a, PE-conjugated IgG1 and PE-conjugated IG2A, purchased from R&D systems (Minneapolis, MN, USA). The following recombinant human interleukins, rhIL-2, rhIL-15, and rhIL-21 (PeproTech, Rocky Hill, NJ, USA), were used to expand the NK cells. Vita-Orange Cell Viability Reagent (WST-8; Biotool, Houston, TX, USA) was used for the cytotoxicity assay. Matrigel (BD Biosciences, San Jose, CA, USA), the reconstituted basement membrane matrix, was used for inducing MDA-MB-231 tumor growth in NSG mice. The use of animals for this study was approved by the Institutional Animal Care and Use Committee of Chonnam National University.

Flow cytometric analyses were performed using a FACS Canto II cytometer (BD Biosciences, San Jose, CA, USA) to assess the purity and CD137-mediated expression of NK cells. The following mAbs were used for staining human peripheral blood mononuclear cells (PBMCs): phycoerythrin (PE)-conjugated CD56, PE CD137L, PE NKp30, PE NKp44, PE NKp46, PE CD122, PE CD117, PE TRAIL, PE CD107a, PE CD158e, PE CCR5, PE CD25, PE CD226, PE NKG2D, PE FasL, PE NKG2A, PE NKG2C, fluorescein isothiocyanate (FITC)-conjugated CD3, FITC CD158a, FITC CD158b, allophycocyanin (APC)-conjugated CD137, APC anti-HER2, APC anti-epidermal growth factor receptor (EGFR), and 7-aminoactinomycin D (7-AAD); all mAbs were obtained from BD Biosciences. Stained cells were collected on a FACS Canto II cytometer (BD Biosciences), and data were analyzed using FlowJo version X software (FlowJo, LLC, Ashland, OR, USA).