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.
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.
1. RAW 264.7 Cell Culture
2. Pathogenic Salmonella enterica Serovar Typhimurium 14028 Transformation and Culture
3. Preparation of Nanoparticle Suspension
4. Phagocytosis Assay
Tips & notes:
5. Sample Acquisition on the ImageStreamX
To summarize, samples can be run in the following order:
Tips & notes:
6. Image Analysis
Tips & notes:
7. Representative Results
The representative images in Figure 2 demonstrate that MIFC can be used to successfully distinguish between internalized (left panel) versus surface bound (right panel) NP (Figure 2A) or Salmonella (Figure 2B). Internalization of NP and Salmonella were reduced by both inhibiting actin and lowering the temperature to 4 °C (Fig 3A). The percentage of cells positive for surface bound NP increased from approximately 8% at 37 °C to greater than 35% after either cytochalasin-D or 4 °C treatment (Fig 3B). In contrast, the percentage of cells with surface bound Salmonella was reduced from 35% to 15% following cytochalasin-D treatment. Incubation of RAW 264.7 cells with Salmonella at 4 °C decreased internalization without an apparent increase in the amount of surface bound bacteria as compared to the 37 °C control. Together, these data demonstrate that Salmonella and NP are internalized by a similar cellular process that requires actin and is temperature dependent. Moreover, the data indicate that sustained attachment of Salmonella to macrophages requires actin polymerization.
Figure 1. Schematic of the gating strategy utilized to determine internalized versus surface bound nanoparticles and Salmonella. (A) To limit the analysis to single cells, it is important to eliminate debris and multi-cellular events. Single cells and doublets were separated from multicellular aggregates using the IDEAS features area and aspect ratio of the brightfield image (M01). Area is the size of the image in square microns and aspect ratio is the minor axis divided by the major axis and therefore a measure of circularity (a perfect circle will have an aspect ratio of 1; doublets typically have aspect ratios of around 0.5 and multicellular aggregates are typically less than 0.5). A region was drawn to gate on single cell events (Step 6.6). (B) To gate on cells in-focus, the IDEAS feature Gradient RMS of the brightfield image is plotted in a histogram. The Gradient RMS feature measures the sharpness quality of an image by detecting changes of pixel values in the image. A higher Gradient RMS value indicates a more focused image (Step 6.7). (C) Green fluorescence positive cells were selected by gating on the cells with high Max Pixel values and Intensity in the green fluorescence channel (Step 6.8). (D) Cells with internalized NP or Salmonella were selected by choosing the cell population with an internalization score equal to or greater than 0.3. Cells receiving a score less than 0.3 were considered to be surface bound (Step 6.9). (E) Cells in the “internalized” gate were further characterized based on the number of spots (NP or STM) because there were some cells with background staining that were counted as internalized but had a spot value of zero (Step 6.11). Representative images are presented for each gate. Click here to view larger figure.
Figure 2. Cell images of internalized and surface bound nanoparticles (NP) or Salmonella. (A) Representative images of RAW 264.7 cells that internalized NP (left panel) and cells to which NP were bound to their surface but not internalized (right panel). (B) Representative images of RAW 264.7 cells that internalized Salmonella (left panel) and cells to which Salmonella were bound to their surface but not internalized (right panel).
Figure 3. Cytochalasin-D treatment of cells inhibited internalization of NP and Salmonella. (A) Pretreatment of RAW 264.7 cells with cytochalasin-D or incubation at 4 °C reduced the incidence of NP or Salmonella internalization as compared to RAW 264.7 cells incubated at 37 °C in medium. RAW 264.7 cells incubated in medium containing DMSO (i.e., vehicle control) showed similar internalization levels for NP and Salmonella compared to medium alone (data not shown). (B) Pretreatment of RAW 264.7 cells with cytochalasin-D increased the percent of cells with surface bound NP while decreasing the percent of cells with surface bound Salmonella.
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 and flow cytometry have been used for quantifying particle uptake; however, their respective limitations with high throughput and resolution call for alternative approaches to study internalization. In this article, we perform MIFC analysis to characterize and compare how the biological process of phagocytosis differs between two types of targets- a bacterial pathogen and synthetic nanoparticles.
The MIFC phagocytosis assay was used to delineate differences in the mechanism underlying the uptake of Salmonella compared to nanoparticles. It should be noted that inhibitors can be cytotoxic depending on their incubation time and concentration; hence a prior cytotoxicity profiling (i.e., exposure time and concentration) is necessary before their use13,14. Depending on their chemical formulation, nanoparticles can have a glass transition temperature (Tg = 13 °C) below RT, consequently, they form aggregates at RT15. To overcome the aggregation, we sonicated the particles and kept them on ice prior to addition to the cell cultures. Kinetic studies of nanoparticle uptake provide important information on the efficiency of these particles to deliver the encapsulated payload to antigen presenting cells. Care must be taken to keep cells on ice at the end of a given time point to inhibit further cellular processes. To this end, we have noted variability in cellular uptake when cells were not placed on ice. One limitation of the method described above is that the experiment is performed on adherent cells that require scraping techniques to harvest the phagocytic cells. This procedure could result in cell death, thus introducing some error in the data analysis. The procedure we describe herein could also be performed using cells that grow in suspension.
The incubation of macrophages with reactive dyes or immunolabeling reagents was optimized by reducing the amount of residual buffer present following fixation. The presence of residual buffer creates a dilution effect and can produce sample to sample variation among the multiple samples, thereby resulting in inconsistent mean fluorescence intensity values. Sample concentration (i.e., cells/mL) is also an important factor to consider during the acquisition step, as dilute samples result in longer run times. For a multi-parametric comparison between different experimental samples, it is important to keep the laser intensity constant.
The authors have nothing to disclose.
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.
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 |