Apc conjugated igg2a

Phycoerytherin (PE) conjugated mouse anti-human CXCR6 antibody, Allophycocyanin (APC) conjugated rat anti-human CXCL16 and their isotype controls PE-conjugated IgG2b, APC-conjugated IgG2a were purchased from R&D Systems (Minneapolis, MN). Culture media from OvCa (SKOV-3 and OVCAR-3) and IOSE-7576 cells was removed and cells were washed with PBS and harvested using Accutase (Corning), subsequently washed with FACS buffer (2% FBS in PBS). 10⁵ cells were treated with Fc Block (PharMingen) for 15 minutes at RT. Then cells were stained with PE-conjugated mouse anti-human CXCR6 and APC-conjugated rat anti-human CXCL16 on ice for 40 minutes. Controls were stained with PE and APC-conjugated isotype controls. After staining unbound antibody was washed with 500 μl FACS buffer and resuspended in 500 μl of 1X fixation buffer (Invitrogen, Frederick, MD) on ice for 10 min. Subsequently, cells were washed and resuspended in FACS buffer. Flow cytometry analysis of CXCR6 and CXCL16 was performed by acquiring 10,000 events using Guava EasyCyte (millipore). Analysis was done using FlowJo software.