Analysis of Macrophage Receptor Interactions with Pathogenic Bacteria Using Flow Cytometry

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 the procedure for labeling host immune receptors that bind bacterial surface antigens, enabling their detection through flow cytometry.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

Mycobacterium tuberculosis-SLAMF1 (Signaling lymphocytic activation molecule 1) interaction by flow cytometry

1. Proteins-bacteria cross-linking
   1. Incubate 1 × 10⁶ whole inactivated Mycobacterium tuberculosis cells (WCMtb, M. tuberculosis inactivated by gamma irradiation) with protein extract from 1 x 10⁶ macrophages (50 µL) overnight at 4 °C in a rotating microtube holder.
      NOTE: 10 µL of WCMtb = 1 × 10⁶ bacteria, quantified by O.D. 600nm in a spectrophotometer, considering an O.D. of 1 as 1 x 10⁸ bacteria/mL.
   2. The following day, add 500 µL of 1% formaldehyde diluted in 1x PBS (phosphate-buffered saline). Incubate for 15 min under agitation (shaker) at room temperature (RT).
   3. Add 25 µL of 0.125 M glycine diluted in water. Incubate for 5 min under agitation (shaker) at RT.
   4. Wash twice with 1 mL of 1x PBS at 14,000 x g for 5 min. Discard the supernatant between washing steps.
   5. Resuspend the pellet and incubate the suspension in 500 µL of 2 mM of ethylene glycol bis (succinimidyl succinate) (EGS) crosslinker diluted in a 1:1 mixture of glacial acetic acid:water for 1 h at RT.
      NOTE: The preparation of the reagents at the time of use is highly recommended. To prepare the EGS, glacial acetic acid, and water must be mixed in equal proportions and heated to 70 °C. If the solution is not hot, the EGS is not completely resuspended.
2. SLAMF1 staining
   1. Repeat step 1.1.4 using 500 µL of 1x PBS in the first wash and 1 mL in the second.
   2. Add an anti-human SLAMF1 antibody to the microtube and vortex it to stain the protein-bacteria complex for 30 min at 4 °C in the dark. Use the amount of antibody suitable for 1 x 10⁶ cells (2.5 µL or 0.125 µg).
   3. Wash the cells to eliminate the excess of antibodies using FACS (Fluorescence-activated cell sorting), resuspend the pellet in FACS, and acquire the sample in a flow cytometer.
      NOTE: Perform the corresponding isotype or FMO (fluorescence minus one) control

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 FITC antibody	eBioscience	11-1509-42	For flow cytometry
anti-SLAMF1 PE antibody	BioLegend	306308	For flow cytometry
anti-SLAMF1 primary antibody	BioLegend	306302	For fluorescence microscopy
Aqua-Poly/Mount	Polysciences	18606-20	Mounting media
CD14 MicroBeads	Miltenyi Biotec	130-097-052	For monocytes isolation
Coverslips 12mm	HDA	-	For interaction assay by microscopy
EGS	ThermoFisher Scientific	21565	For crosslinking treatment
FACSCanto II	BD Biosciences	338960	Flow cytometer with BD FACSDiva software
Fetal Bovine Serum	Natocor	-	Inactivated and irradiated, for macrophages culture
Ficoll-Paque PLUS	Cytiva	17144003	For PBMCs separation
Fiji/ImageJ	Open Source software	-	For micrographs analysis
FlowJo 7.6.2	Tree Star	-	For flow cytometry analysis
Formaldehyde	Merck	K47740803613	For crosslinking treatment
Glass slides	Glass Klass	-	For interaction assay by microscopy
Glycine	Sigma	G8898	For crosslinking treatment
M. tuberculosis, strain H37Rv, gamma-irradiated whole cells	BEI Resources, NIAID, NIH	NR-14819	For interaction assay
M. tuberculosis, strain H37Rv, whole cell lysate	BEI Resources, NIAID, NIH	NR-14822	For macrophages stimulation and interaction assay
Neofuge 13R	Heal Force	Neofuge 13R	High Speed Refrigerated Centrifuge for protein extraction
Penicillin/Streptomycin	Gibco	15140122	For macrophages culture
PMSF	ThermoFisher Scientific	36978	For proteins isolation
Poly-D-Lysine	Sigma Aldrich	A-003-M	For coverslips treatment
Protease Inhibitor Cocktail	Sigma Aldrich	P8340	For proteins isolation
RPMI 1640	Gibco	11875093	For macrophages culture
Sorvall ST 16/16R centrifuge	ThermoFisher Scientific	75004240	For PBMCs and monocytes isolation