A method permissive to fixation and permeabilization for the multiparametric analysis of apoptotic and necrotic cell phenotype by flow cytometry. Issue 11 (26th October 2017)

• Record Type: Journal Article
• Authors: Mariotti, Sabrina; Pardini, Manuela; Teloni, Raffaela; Gagliardi, Maria Cristina; Fraziano, Maurizio; Nisini, Roberto
• Is Part Of: Cytometry. Volume 91:Issue 11(2017)
• Page Start: 1115
• Page End: 1124
• Publication Date: 2017-10-26
• Subjects: apoptosis -- necrosis -- pyroptosis -- Mycobacterium tuberculosis -- fixation -- intracellular staining; Flow cytometry -- Periodicals; Imaging systems in biology -- Periodicals; Imaging systems in medicine -- Periodicals; Diagnostic imaging -- Periodicals; 571.605
• DOI: 10.1002/cyto.a.23268
• Languages: English
• ISSNs: 1552-4922

Abstract

Abstract: Annexin‐V/propidium iodide method (A‐V/PI) is a common flow cytometric method for the multiparametric analysis of cells in apoptosis. However, A‐V/PI does not permit fixation and/or permeabilization of cells making impossible evaluation of intracellular markers, restricting the analysis in a narrow time frame after staining and excluding the possibility to study pathogen‐infected cells. We developed a method permitting fixation and permeabilization of stained cells: Fixed Apoptotic/Necrotic (FAN) cells test. FAN relies on the same principle of A‐V/PI, but uses reagents that maintain their binding and fluorescence characteristics after fixation/permeabilization: a fluorochrome‐labeled anti‐phosphatidylserine antibody and fluorescent amine‐binding dyes. FAN was tested to discriminate apoptotic and necrotic cells using different stimuli on several cell types and results were always comparable to those obtained using A‐V/PI. FAN, unlike A‐V/PI, permitted to correlate cell death with intracellular and surface markers expression and to perform cytometry even two weeks after sample preparation. As fixation of stained cells inactivates infective pathogens, we used FAN in an in vitro model of Mycobacterium tuberculosis (Mtb) infection of macrophages to monitor cellular infection and cell death induction. Using a red‐fluorescent Mtb, fluorochrome labeled anti‐TNF‐α and anti‐MHC class II monoclonal antibodies, FAN permitted to establish that the extent of macrophage death correlates with intracellular Mtb content and that dying cells accumulate TNF‐α and down‐modulate MHC class II molecules. Results suggest that FAN may represent an additional tool to study programmed cell death particularly useful when fixation procedures are required for a safe infected sample analysis or to comparatively analyze multiple samples. © 2017 International Society for Advancement of Cytometry