This study investigates the process of transinfection between dendritic cells and CD4 + T cells using a specific peptide. The experimental setup includes both test and control wells to analyze the interactions and effects of bacterial infection on T cell activation.
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 polycarbonate trans-well barriers with a three micrometer pore size into the third and fourth wells. To the first well, add 0.1 milliliters of the infected overloaded dendritic cells. To the second well, add 0.1 milliliters of the infected non-loaded dendritic cells.
Add 0.1 milliliters of the infected overloaded dendritic cells to one well, and 0.1 milliliters of the infected non-loaded dendritic cells into the other well. Finally, as a negative control, directly infect the fifth well of CD4+ T cells at an MOI of 10. Then, bring the total volume of the well up to 0.6 milliliters with RPMI.
Incubate the plate for 30 minutes to allow immune synapses to form. After the incubation, add 100 micrograms of Gentamicin per milliliter of medium to each well, and incubate for one hour. Gentamicin is an aminoglycoside antibiotic that is not able to penetrate eukaryotic cells.
Therefore, in this step, aminoglycoside will kill extracellular bacteria but not intracellular bacteria.
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