In Vitro Bacterial Transinfection of T Cells via Immunological Synapse Formation

Begin with wells containing CD4⁺ T cells expressing receptors specific to the ovoalbumin peptide or OVAp.

Into the negative control well, place a porous membrane insert.

In a tube, take dendritic cells infected with bacteria and loaded with OVAp for surface presentation.

Add dendritic cells directly into the test well and into the insert in the negative control well.

Add media to the wells and incubate.

In the test well, cells interact to form an immunological synapse, facilitating bacterial transfer from the dendritic cell to the T cell, a process known as transinfection.

In the negative control well, the membrane barrier prevents cell-cell interaction, thereby inhibiting transinfection.

Add the antibiotic gentamicin to the wells and incubate.

Gentamicin cannot penetrate eukaryotic cells, thus killing only non-internalized bacteria while internalized bacteria survive.

The test sample containing infected T cells and the control sample with uninfected T cells are now ready for T cell isolation and transinfection quantification.

To a 24-well culture plate, add two million CD4⁺ T cells in a volume of 0.5 milliliters to each of five wells.

Next, to control for physical contact, place polycarbonat