Percp cy5.5 conjugated anti human cd3

Subjects included in this study were undergoing percutaneous liver biopsy for diagnostic purposes. A cell strainer (FALCON, 352350) was used to mash the biopsy samples. After that, the samples were digested by collagenase (0.05%)/Hyaluronidase (1000 U/mL) and DNase (5 U/mL) to obtain a cell suspension. Subsequently, the supernatant was removed, and the pellet was treated with an RBC lysis buffer as previously described (19 (link)).
Multicolor flow cytometry was performed using the following commercially available antibodies: phycoerythrin (PE)-conjugated anti-human CD38 and CD80, PerCP-Cy5.5-conjugated anti-human CD16, PE-cyanine 7 (Cy7)-conjugated anti-human NKG2D (BioLegend, San Diego, CA, USA), allophycocyanin (APC)-Cy7-conjugated anti-human HLA-DR, APC-conjugated anti-human CD69, V450-conjugated anti-human CD8, V500-conjugated anti-human CD4, and PerCP-Cy5.5-conjugated anti-human CD3 (BD Biosciences, San Jose, CA, USA). LIVE-DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific, L34955) was used to discriminate dead cells from live cells. Data were recorded using an LSR Fortessa, Canto II instrument (BD Biosciences) and analyzed using the FlowJo software (TreeStar, Ashland, OR, USA). Intracellular cytokine staining was performed using a previously described protocol (20 (link)).

HCT116, NCI-H630 and RKO cells were cultured in RPMI 1640 medium supplemented with 10 % FBS, 2 mM Ultraglutamine and 100U/ml penicillin/streptomycin (Lonza, BioWhittaker). The packaging cell line Phoenix-A and HEK 293 T cells were cultured in DMEM medium (Lonza, BioWhittaker) supplemented with 10 % FBS, 2 mM Ultraglutamine and 100U/ml penicillin/streptomycin. TNF-α and IFN-γ (Peprotech) were used for in vitro stimulation of cell lines.
Monoclonal antibodies used in this study were as follows: anti-CD3 mAB OKT3 (EB16-0037-85, eBioscience), PE-conjugated anti-human CX3CR1 (D070-5, MBL), allophycocyanin (APC) conjugated anti-human CD3 (555342, BD Biosciences), APC-Cy7 conjugated mouse anti-human CD8 (557852, BD Biosciences), PerCP-Cy5.5-conjugated anti-human CD3 (560835, BD Biosciences), APC conjugated anti-human CD45 (560973, BD Biosciences), PE-Cy7 conjugated anti-human CD4 (557852, BD Biosciences). Other reagents included RetroNectin (T100-A, Takara); recombinant human IL-2 (202-IL, R&D Biosystems); Collagenase Type I (C0130, Sigma); DNAse I (D5025, Sigma), Hyaluronidase (H3506, Sigma).