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

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

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

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

Hight Throughput, Parallel Imaging and Biomarker Quantification of Human Spermatozoa by ImageStream Flow Cytometry

8 páginas, 2 figuras, 1 tabla.Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.Peer reviewe

Detection of CD169⁺ IL-7Rα^hiCCR6⁺ lymphocytes in digested lymph node tissue.

<p>(A) Flow cytometric detection of IL-7Rα expression on CD169⁺CD11c^lo cells from digested lymph nodes. (B) Immunofluorescence microscopy of a lymph node section stained with anti-CD169 and anti-IL-7Rα monoclonal antibodies. Enlargements (bottom, far right) show examples of IL-7Rα⁺ cells (white arrows) closely associated with CD169⁺ SSMs. FO, follicle; T, T zone. (C) Flow cytometric analysis showing CD169⁺CD11c^lo cells contain a population of IL-7Rα^hiCCR6⁺ cells. All data in A–C are representative of at least three independent experiments. (D) Flow cytometric analysis of digested lymph node cells from a control and TCRβδ-deficient mouse. IL-7Rα^hiCCR6⁺ cells gated from total cells express CD169 on a fraction of both CD3e⁺ and CD3e⁻ cells. Data are representative of at least three independent experiments (control mice) and one experiment in which a TCRβδ-deficient mouse was analyzed.</p

Analysis of CD169 distribution on IL-7Rα^hiCCR6⁺ lymphocytes and CD11c^hi dendritic cells using ImageStreamX imaging flow cytometry.

<p>(A) Gating scheme to identify CD169⁺IL-7Rα^hiCCR6⁺ cells analyzed on an ImageStreamX imaging flow cytometer (Amnis Corp). The area of CD169 staining on images of the gated cells was quantified using a Threshold mask on the upper 60% of the pixel intensities (right panel). (B) Representative images of cells with small, medium, and large areas of CD169 area based on the histogram in (A). Channels for CD169 (green), IL-17Rα (red), and brightfield are shown. Graph on right shows the frequency of cells falling in each gate (n = 2 mice, lines indicate means). (C) Gating scheme to identify CD169⁺CD11c⁺ cells analyzed on an ImageStreamX imaging flow cytometer. The area of CD169 staining on images of the gated cells was quantified as described in (A). (D) Representative images of cells with small, medium, and large areas of CD169 area based on the histogram in (C). Channels for CD169 (green) IL-17Rα (red), and brightfield are shown. Graph on right shows the frequency of cells falling in each gate (n = 2 mice, lines indicate means). Data are representative of one experiment with two mice. In a second experiment with two mice, CD169⁺ blebs were visualized on CD169⁺CCR6⁺TCRγδ⁺ cells.</p

Analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry.

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells. 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. 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.This is an article from Journal of Visualized Experiments 64 (2012): e3884, doi: 10.3791/3884.</p