Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

ANGELA MARIA BARBERO; HERNÁNDEZ DEL PINO RODRIGO E; , GARCÍA V.E; PASQUINELLI VIRGINIA

Journal of Visualized Experiments

Journal of Visualized Experiments

Año: 2025

1940-087X

The evaluation of direct interaction between pathogens and immune receptorsusually involves sophisticated techniques or implies the use of transgenic strainsand genetically engineered cells. Here, an alternative method to detect biochemicalinteraction between the macrophage microbial sensor SLAMF1 and Mycobacteriumtuberculosis is described. Two technical approaches employing flow cytometryand fluorescence microscopy were developed. Total cell protein extracts fromhuman macrophages were generated, then incubated with whole cells of M.tuberculosis (WCMtb) or M. tuberculosis antigens (Mtb Ags) overnight at 4 °Cand finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidylsuccinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry wasdetected with a PE-specific anti-SLAMF1 antibody. The existence of interaction byfluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Agsto poly-D-lysine coated slides, which were incubated with the total protein extractfrom monocyte-derived macrophages. After cross-linking treatment, SLAMF1 wasvisualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies.The assays provided a strong biochemical tool to measure pathogen-immunoreceptorinteractions, overcoming the difficulties associated with transgenic cell lines andprotein gene expression modulation experiments.