Fluorescence-Based Assay for Investigating Macrophage Receptor Interactions with Bacterial Antigens

Abstract

Source: Barbero, A. M., et.al. Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1. J. Vis. Exp. ( (2025)

This video demonstrates a fluorescence-based assay to study the interaction between a macrophage receptor and Mycobacterium tuberculosis antigens. Fluorescently labeled antigens on a coverslip are incubated with macrophage protein extracts, cross-linked, antibody-labeled, and visualized by overlapping fluorescence signals.

Protocol

1. Mycobacterium tuberculosis -SLAMF1 interaction by fluorescence microscopy

1. Bacteria-protein cross-linking
   1. Introduce 12 mm round coverslips into 24-well culture plates (1 coverslip per well) using round-nosed surgical tweezers.
      NOTE: Clean the coverslips thoroughly with 70% ethanol. If desired, they can be autoclaved or sterilized under UV light for 30 min.
   2. Incubate the coverslip with 400 µL of 10 µg/mL Poly-D-Lysine overnight at 4 °C. Cover the plate with aluminum foil to protect it from light.
   3. The next day, re....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488 secondary antibody	Invitrogen	A21121	For fluorescence microscopy
Anti-SLAMF1 primary antibody	BioLegend	306302	For fluorescence microscopy
Aqua-Poly/Mount	Polysciences	18606-20	Mounting media
Coverslips 12mm	HDA	-	For interaction assay by microscopy
EGS	ThermoFisher Scientific	21565	For crosslinking treatment
Formaldehyde	Merck	K47740803613	For crosslinking treatment
Glass slides	Glass Klass	-	For interaction assay by microscopy
Glycine	Sigma	G8898	For crosslinking treatment
Imager.A2	Carl Zeiss	430005-9901-000	Fluorescence microscope with Colibri 7 illumination module
Poly-D-Lysine	Sigma Aldrich	A-003-M	For coverslips treatment
Rhodamine B	Sigma Aldrich	21955	For M. tuberculosis staining