Phenotyping EBV-Infected Cell Lines Using Flow Cytometry

Take lymphoid cell lines established from Epstein–Barr virus (EBV)-infected patients.

These cells express oncogenic viral proteins that activate signaling pathways, promoting immortalization and a chronically activated state marked by HLA-DR expression, a molecule involved in antigen presentation.

Centrifuge the tubes and wash the cells with a buffer.

Add serum to block non-specific binding sites. Centrifuge the cells, and add the buffer.

Divide each suspension into tubes, then centrifuge.

To each tube, add a unique pair of fluorescently labeled antibodies, one tagged with a green fluorophore and the other with a red, each targeting specific surface markers.

Centrifuge the tubes, add the buffer to wash the cells, and resuspend them.

Use flow cytometry to detect fluorescence from the markers.

Both lines express HLA-DR, indicating EBV-driven immune activation, but differ in lineage-specific markers that confirm one as a T cell and the other as an NK cell line.

When the cells have expanded, collect one times ten to the sixth cells to determine the phenotypes of the cell lines. Centrifuge them at 240 times g for five minutes at room temperature to precipitate the NKNT cells.

After that, discard