Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Abstract

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells [1][2][3][4][5] . Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes 6-8 . Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStream X system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis. Video LinkThe video component of this article can be found at http://www.jove.com/video/3884/ Protocol 1. RAW 264.7 Cell Culture 1. Harvest RAW 264.7 cells from their flasks when they reach confluency by scraping them gently with a cell scraper. Count and plate them into a 24-well cell culture dish at a density of 5 x 10 5 cells/well in 0.5 mL complete Dulbecco's Modified Eagle Medium (cDMEM; 10% heat-inactivated fetal bovine serum (FBS), 2 mM Glutamax, and 10 mM HEPES) and incubate overnight at 37°C in a 5% CO2 incubator. Pathogenic Salmonella enterica Serovar Typhimurium 14028 Transformation and Culture1. Introduce recombinant plasmids expressing green fluorescent protein (GFP) to S. Typhimurium by electroporation. First, prepare culture of Salmonella enterica serovar Typhimurium (ATCC 14028) in LB broth and incubate overnight at 37°C with aeration. 2. Next day, pellet 1 mL of bacteria at maximum speed in a microcentrifuge. Wash the cells four times with 1 mL of cold MOPS/glycerol solution (1 mM 3-(N-morpholino)propanesulfonic acid (...

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood

“…Nonetheless, flow imaging based assays, similar to conventional flow cytometric assays, are able to incorporate additional read outs such as staining for markers of immune activation and function thereby allowing direct associations to be made between TiO 2 interactions with single cells (adhesion or internalization) and the functional consequences of such interactions on the same cell. Like others, we found imaging cytometry a powerful technique for the analysis of particle localization, this was dependant on tailoring custom masks in order to obtain an accurate analysis 9, 10, 11, 12, 13, 14, 15. There is always potential for some particles analyzed to be attached externally to the front or back of the cells, the occurrence of false positive internalization events is random and at the same frequency within all data files as it is dependent only on the orientation of the cells as they are imaged.…”

An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry

“…Therefore, our studies were carried out at low particle:cell ratios for maximal accuracy. However, in studies that used other bacterial species or particle types such as nanoparticles and exosomes, larger numbers of particles per cell could be accurately counted (Phanse et al, 2012; Franzen et al, 2014). We advise that researchers keep the particle;cell ratio as low as possible and empirically determine an appropriate cutoff for the spot count algorithm for each experimental condition, based on properties of the particles and cells.…”

“…To date, imaging flow cytometry has been used in several studies examining internalization of nanoparticles, exosomes, and bacteria (Ploppa et al, 2011; Phanse et al, 2012; Vranic et al, 2013; Franzen et al, 2014). In these studies, internalization was quantified by quantifying fluorescence in the cells within the whole cell mask, eliminating signal coming from the particles at the periphery of the mask.…”

Anti-inflammatory Chitosan/Poly-γ-glutamic acid nanoparticles control inflammation while remodeling extracellular matrix in degenerated intervertebral disc

“…Internalization quantification is described in Supplementary Materials and Methods (S1.1). Briefly, internalization score was calculated based on the ratio of the FITC fluorescence intensity inside the cell and the intensity of the entire cell [34,35]. Higher scores denote larger NPs concentration in the cell cytoplasm, while negative scores denote cells with little internalization.…”