Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry
Summary
In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.
Abstract
Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. 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 sizes6-8. 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 ImageStreamX 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.
Protocol
Discussion
Studies have shown that biodegradable nanoparticles based on poly(lactic-co-glycolic acid (PLGA) or polyanhydrides can be used to deliver encapsulated antigens or drugs to target cells. Uptake of these nanoparticles by phagocytic cells is important for their effectiveness, thus making quantitative analysis of internalization critical in designing novel nanoparticle delivery systems. By using this method, differential uptake of nanoparticles by various cell types can be analyzed with ease. To date, conventional microscopy…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank the ONR-MURI Award (NN00014-06-1-1176) and the U.S. Army Medical Research and Materiel Command (Grant Numbers W81XWH-09-1-0386 and W81XWH-10-1-0806) for financial support.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| RAW 264.7 cell line | American Type Culture Collection (ATCC) | TIB-71 | |
| Dulbecco’s Modified Eagle Medium (DMEM) | Cellgro | 10-013-CV | |
| Fetal bovine serum | Atlanta Biologicals | S 11150 | Premium Grade |
| Glutamax | Gibco | 35050-061 | |
| HEPES | Gibco | 15630-080 | |
| 24-well plate | TPP | 92024 | |
| Cell culture Flasks | TPP | 90151 | |
| Cell scraper | TPP | 99002 | 24 cm |
| Salmonella entericaserovar Typhimurium | ATCC | 14028 | |
| BTX ECM630 Electro Cell Manipulator | BTX Harvard Apparatus | ||
| MOPS | Fisher Scientific | BP308 | |
| Phosphate buffered saline (PBS) | Cellgro | 21-040-CV | |
| Ultrasonic liquid processor | Misonix | S-4000 | |
| Cytochalasin-D | Sigma-Aldrich, | C8273 | |
| Formaldehyde | Polysciences | 04018 | |
| Wash buffer | 2% heat inactivated FBS, 0.1% sodium azide in PBS. | ||
| Perm/wash buffer | BD Biosciences | 554714 | |
| Clear-view snap cap microtubes | Sigma | T4816 | |
| Alexa Fluor phalloidin 660 | Invitrogen | A22285 | |
| ImageStreamX | Amnis Corporation | 100200 | Options: 658nm laser, autosampler |
| Sodium azide | Fisher Scientific | S 227I-500 |
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