Analysis of Macrophage Receptor Interactions with Pathogenic Bacteria Using Flow Cytometry

الملخص

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.

البروتوكول

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 o....

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
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