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 cells^1-5. 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.

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 10⁵ 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.

2. Pathogenic Salmonella enterica Serovar Typhimurium 14028 Transformation and Culture

1. Introduce recombinant plasmids expressing green fluorescent protein (GFP) to....

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.......

Materials

List of materials used in this article

Name	Company	Catalog 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, by Life Technologies	35050-061
HEPES	GIBCO, by Life Technologies	15630-080
24-well plate	Techno Plastic Products	92024
Cell culture Flasks	Techno Plastic Products	90151
Cell scraper	Techno Plastic Products	99002	24 cm
Salmonella entericaserovar Typhimurium	ATCC	14028
BTX ECM630 Electro Cell Manipulator	BTX Technologies
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, Inc.	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-Aldrich	T4816
Alexa Fluor phalloidin 660	Invitrogen	A22285
ImageStreamX	Amnis Corporation	100200	Options: 658nm laser, autosampler
Sodium azide	Fisher Scientific	S 227I-500