Analysis of Macrophage Receptor Interactions with Pathogenic Bacteria Using Flow Cytometry

Begin with a tube of macrophage protein extract containing host immune receptors.

Add inactivated pathogenic bacteria to provide surface antigens specific to pathogenic strains.

Incubate with rotation to enable immune receptors to bind bacterial antigens, forming receptor–antigen complexes.

Add a crosslinking agent and incubate with shaking to stabilize receptor–antigen complexes.

Introduce a quenching agent to stop the crosslinking reaction.

Add buffer and centrifuge to pellet the complexes.

Remove excess reagents, then resuspend the pellet in a second crosslinking agent to further stabilize the complexes.

Centrifuge and wash the pellet with buffer.

Add fluorescently labeled antibodies and vortex.

Incubate under cold, dark conditions to allow antibodies to bind receptor–antigen complexes.

Resuspend the cells in buffer containing serum.

Finally, analyze receptor–antigen complexes using flow cytometry.

As complexes pass through the laser, they emit fluorescence signals that correlate with receptor-antigen binding, highlighting host-pathogen recognition.

Incubate one times 10 to the power of six whole inactivated Mycobacterium tuberculosis cells with 50 microliters of protein extract from one times ten to the power of six macrophages overnight at 4 degrees Celsius in a rotating microtube holder.

The next day, to stain the protein bacteria complex, add the anti-human SLAMF1 antibody to the microtube, vortex the mixture, and incubate overnight for 30 minutes at 4 degrees Celsius in the dark. After washing the protein bacteria complexes with FACS, resuspend them in FACS buffer and acquire the sample on a flow cytometer.