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 the supernatant, wash the precipitation by adding 7 milliliters of PBS. Centrifuge it for five minutes at 240 times g at room temperature. Repeat the procedure once more. Then, add 200 microliters of human serum to each tube, mix well, and incubate for 10 minutes at room temperature. Centrifuge the cells at 240 times g for five minutes at room temperature.

Discard the supernatant and resuspend the cells at one times ten to the sixth cells per milliliter with cold PBSA. Subsequently, divide the cells into five tubes, each containing two times ten to the fifth cells. After that, centrifuge the cells at 240 times g for five minutes at four degrees Celsius. Discard the supernatant and resuspend the cells with PBSA buffer or PBSA containing 10 microliters of PE and 10 microliters of FITC-labeled antibodies to stain cell receptors of T or NK cells on ice.

Cells suspended with PBSA buffer are used as a negative control. Next, incubate the cells with antibodies for 20 to 30 minutes on ice in the dark. Wash the cells twice with cold PBSA and resuspend the cells with 300 microliters of cold PBSA before the analysis with the flow cytometer.