Name: integrate imaging flow cytometry and transcriptomic profiling to evaluate altered endocytic cd1d trafficking
Uploaded: 2018-10-29T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking at JoVE.com

The authors thank Robert Giulitto (Hoxworth blood center) for human blood samples and Dr. Liang Niu for the normalization of gene expression reads. We also thank grant support from the National Institute of Environmental Health Sciences (ES006096), Center for Environmental Genetics (CEG) pilot project (S.H.), National Institute of Allergy and Infectious Diseases (AI115358) (S.H.), University of Cincinnati University Research Council award (S.H.), and University of Cincinnati College of Medicine Core Enhancement Funding (X. Z.).

Human protocols in this study were approved by the Institutional Review Board of the University of Cincinnati and all methods were performed in accordance with the relevant guidelines and regulations. Blood samples from healthy donors were obtained from the Hoxworth Blood Center at the University of Cincinnati Medical Center.
1. Transcriptomic Profiling of Pollutant-exposed Human Monocyte-derived DCs
<ol>
	<li>Total RNA extraction of BaP-exposed DCs
	<ol>
		<li>Differentiate human DCs using cytokines GM-CSF and IL-4, and expose DCs to BaP for 4 days13.</li>
		<li>Sort BaP-exposed DCs using flow cytometry based on the surface expressed markers (Lineage-HLA-DR+), the majority of which consists of conventional DCs (CD11c+CD1c+)13. Typically, sort 10,000 DCs into culture medium containing 10% FBS, centrifuge DCs at 600 x g and 4 &#176;C for 6 min, and immediately remove the supernatant by aspiration using a 1 mL pipette tip.</li>
		<li>Once the supernatant is removed by aspiration, lyse the cell pellet by adding 0.4 mL of Lysis/Binding Buffer from the RNA isolation kit for storage in -80 &#176;C before RNA extraction.</li>
		<li>Use the RNA isolation kit with the total RNA extraction protocol to extract the total RNA14. Measure the RNA integrity using a Bioanalyzer15.</li>
	</ol>
	</li>
	<li>Transcriptomic analysis of sorted DCs (Figure 1A)
	<ol>
		<li>Prepare the library for RNA-seq using a RNA Library preparation kit. In short, fragment the isolated poly-A RNA (~200 bp), reverse transcribe the fragments to the 1st strand of the cDNA, and follow by the second strand cDNA synthesis labeled with dUTP.</li>
		<li>Ligate double strand cDNA to the adapter with a stem-loop structure after bead purification, end-repairing and dA-tailing. Excise the uracil residues in the 2nd strand of the cDNA and adapter loop with USER (Uracil-Specific Excision Reagent) enzyme to maintain strand specificity and open the adapter loop for PCR.
		<ol>
			<li>Perform 13 cycles of PCR using universal and index-specific primers to enrich the indexed library. Clean up the library and run on a Bioanalyzer DNA High Sensitivity chip to check the library quality and size distribution.</li>
		</ol>
		</li>
		<li>Use a library quantification kit and a real-time PCR system combined with library size information to&#160;calculate library concentration via a standard curve method.&#160;</li>
		<li>Proportionally&#160;pool individually indexed compatible libraries and adjust the final total concentration to 15 pM. Perform library cluster generation and sequence the library at a setting of single read 1x 50 bp to&#160;generate ~25 million reads per sample.</li>
	</ol>
	</li>
	<li>Sequencing
	<ol>
		<li>Load the sequencing and indexing reagents to the SBS and PE reagent racks, respectively. Place the reagents in a laboratory-grade water bath for 1 h until all the ice has melted and the reagents in each bottle/tube are mixed properly.</li>
		<li>Prepare the ICB mix by adding the thawed dye and -20 &#176;C enzyme to the bottle and mix. Prepare a NaOH solution according to the sequencing instructions. Place all reagents at 4 &#176;C until ready to use.</li>
		<li>During the 1 h waiting period, power on the sequencer. Wait for the DONOTEJECT drive to appear and connect the computer to a network drive.</li>
		<li>Launch the sequencer control software.</li>
		<li>Prepare 2 L of Maintenance Wash solution that contains 0.5% Tween 20 and 0.03% ProClin 300 in laboratory-grade water.</li>
		<li>In the SBS reagent rack, add ~100 mL of Maintenance Wash solution to each of the 8 bottles, and screw funnel caps to the bottles. In the PE reagent rack, add ~12 mL of Maintenance Wash solution to each of the ten 15 mL conical tubes, and discard the caps.</li>
		<li>Load the two racks with the solution filled bottles/tubes to the sequencer.</li>
		<li>From the sequencer control software, choose the Maintenance Wash; follow the instructions on screen to clean the sequencer fluid system until the process is completed.</li>
		<li>Start a New Run in the &#34;SEQUENCE&#34; tab from the software; direct the output data to a network drive. Choose parameters for single read 1x 50 bp with single index multiplexed libraries.</li>
		<li>Optionally, log into the BaseSpace Sequence Hub so that the sequencing status can be remotely monitored via a computer or smart phone.</li>
		<li>Upload a Sample Sheet for demultiplexing and provide reagent information according to the software requirement.</li>
		<li>Load SBS and PE reagents to the sequencer. Prime the system with a used flow cell (~15 min).</li>
		<li>Once the cluster generation is completed (~4.5 h), take the flow cell out, lightly spray the flow cell with water, and wipe it dry using lens paper. Lightly spray the flow cell with 95% ethanol and wipe it dry. Check against a light to make sure that the surface is clean without debris or salt residue.</li>
		<li>After the Prime step is completed, load the clustered flow cell and start the sequencing. The Sequence Analysis Viewer software will automatically be started.</li>
		<li>Monitor the sequencing data quality via SAV including cluster density, reads pass filter, cluster pass filter %, % &#8805;Q30, Legacy phase/prophase %, indexing QC, etc. This helps to understand the data quality and troubleshoot.</li>
		<li>Change the flow cell gasket and perform a Maintenance Wash after the sequencing is completed. The sequencer is ready for the next run.</li>
	</ol>
	</li>
	<li>Bioinformatic analysis
	<ol>
		<li>Perform bioinformatics RNA-seq data analysis13.</li>
	</ol>
	</li>
</ol>
2. Pathway Analysis of Transcriptomic Profiles (Figure 1B)
<ol>
	<li>Use an edgeR Bioconductor to compare resultant gene expression intensity counts between BaP-exposed and non-exposed DCs from three donors. Then, identify differentially expressed genes between BaP-exposed and non-exposed DCs based on the absolute fold change (&#62;2 folds) and the false discovery rate&#160;(FDR)-adjusted p-values (&#60;0.05).</li>
	<li>To predict the functional clusters of differentially expressed genes, use the ToppCluster software package to search the altered genes against several databases including KEGG and REACTOME and generate clustering data. ToppCluster uses the hypergeometric test to obtain functional enrichment&#160;via the gene list enrichment analysis16.</li>
	<li>Further input the results from these function clusters to Cytoscape17,18 Version 3.3.0, a broadly used open source software platform for visualizing complex networks. Thus, the genes involved in different clusters or pathways, including endocytic clusters and lipid metabolism, are shown in a network with color annotation of the averaged fold change of gene expression (Figure 1B)13.</li>
</ol>
3. Imaging Flow Cytometry of CD1d and Lamp1 Colocalization
<ol>
	<li>Antibody labeling of BaP-exposed DCs
	<ol>
		<li>Expose 0.5 x 106 human DCs to 5.94 &#956;M BaP for 4 days at 37 &#176;C in 2 mL of complete Dulbecco&#8217;s Modified Eagle&#8217;s Medium. Harvest DCs by centrifugation at 400 x g for 10 min. Block the differentiated DCs by incubating cells with human serum blocker and anti-human Fc receptor antibodies for 10 min in ice, including anti-human CD16, CD32, and CD64 antibodies.</li>
		<li>Incubate Brilliant Violet 421-anti-CD1c (L161), phycoerythrin/cyanine dye 7 (PE/Cy7)-anti-HLA-DR, and PE/Cy5-anti-CD11c with DCs for 20 min in ice. Table 1 shows this list of antibodies.</li>
		<li>Fix DCs with 4% paraformaldehyde in PBS and permeabilize them with Permeabilization Wash Buffer. Perform the intracellular staining with a mixture of PE-labeled purified anti-human CD1d (51.1) and&#160;Alexa Fluor 647-labeled anti-Lamp1 (CD107a) (H4A3).</li>
	</ol>
	</li>
	<li>Imaging flow cytometry measurement (Figure 2)
	<ol>
		<li>Analyze the stained samples using an imaging flow cytometer at the flow cytometry core of Cincinnati Children&#8217;s Hospital using a 40X objective to yield 300 pixels for a cell with around 10 &#956;m in diameter. One-pixel size is approximately 0.5 &#956;m by 0.5 &#956;m of the object.</li>
		<li>Acquire 10,000 cellular images in an unbiased manner according to the manufacturer&#8217;s instruction.</li>
	</ol>
	</li>
</ol>
4. Imaging Analyses of Flow Cytometry Images
<ol>
	<li>Colocalization analysis using IDEAS software (Figure 2)
	<ol>
		<li>Perform compensation with single-stained versus non-stained samples by setting the fluorescence intensity of non-stained samples below the threshold, including non-stained BaP-exposed DCs with BaP autofluorescence, as in routine flow cytometry data analysis.</li>
		<li>Gate stained cells to obtain the subsets of HLA-DR+CD11c+CD1d+Lamp1+ cells (Figure 2A) and show cellular images in the gated subsets (Figure 2B).</li>
		<li>Save cell images in a png format based on two technical inclusion criteria, the visual presence of strong co-stained signals and subcellular localization of CD1d and Lamp1 proteins, for the colocalization analyses using ImageJ-Fiji (Figure 2B).</li>
	</ol>
	</li>
	<li>Use software ImageJ-Fiji to perform colocalization analysis (Figure 3).
	<ol>
		<li>Prepare the input image files by merging the 100 saved cell images for non-exposed and BaP-exposed human DCs, respectively (Figure 3A).</li>
		<li>Analyze CD1d (Red) and Lamp1 (Green) colocalization using a scatterplot.
		<ol>
			<li>Run ImageJ-Fiji program. Open the .png file with 100 cell images (Figure 3B and Figure 4A): &#34;File | Open&#34;.</li>
			<li>Split the image with merged red and green channels into two images with either a red or a green channel: &#34;Image | Color | Split channels&#34;.</li>
			<li>Draw a scatterplot using the commands &#34;Analyze | Colocalization | Colocalization Threshold&#34;. Save the scatterplot using the &#34;PrintScreen&#34; key.</li>
		</ol>
		</li>
		<li>Calculate Mander&#39;s colocalization coefficients for each single cellular image (n=100) (Figure 4B)
		<ol>
			<li>Select a single cell image on the image file with split channels using the &#34;Oval&#34; selection tool.</li>
			<li>Use the commands &#34;Analyze | Colocalization | Colocalization Threshold&#34;. Select &#34;Channel 1&#34; from the dialogue box of region of interest (ROI). Keep all calculation options including &#34;Mander&#8217;s using thresholds&#34; for each cell image.</li>
			<li>Repeat this calculation for all 100 cell images.</li>
			<li>Save and open the results using a spreadsheet.</li>
			<li>Plot the average and standard errors for &#8220;thresholded Mander&#8217;s coefficients&#8221; (n=100, 0 means no colocalization and 1 means perfect colocalization). Use Student&#8217;s t-test to calculate the p value for the comparison between BaP-exposed and non-exposed groups (Figure 4B).</li>
		</ol>
		</li>
		<li>Calculate the percent of thresholded pixel intensity co-localized between CD1d and Lamp1 for multiple cell images (n=100) (Figure 4C).
		<ol>
			<li>Use the same analysis protocol in 4.2.3 and additionally select the result option &#34;% intensity above threshold colocalized&#34;.</li>
			<li>Perform this analysis together with Mander&#8217;s colocalization coefficients.</li>
			<li>Similarly plot the average and standard errors for % intensity colocalized between CD1d and Lamp1. Use Student&#8217;s t-test to calculate the p value for comparison between BaP-exposed and non-exposed groups (Figure 4C).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose

Functional confirmation of an altered gene pathway is challenging, because of widely impacted gene expression involving multiple pathways and the difficulty to&#160;integrate individual and populational cellular activities. We employed imaging flow cytometry to specifically test CD1d trafficking in endocytic compartments. Imaging flow cytometry integrates the populational measurement of cells and the individual demonstration of subcellular colocalization of multiple proteins. Confocal microscopy has previously provided h...

Antigen presentation typically involves intracellular protein trafficking, which has been often investigated using morphological characterization and phenotypic profiling of antigen presenting cells1,2,3. To integrate the advantages of imaging and phenotyping methods, we describe an imaging analysis platform at both single cell and population levels to demonstrate an altered protein colocalization in human dendritic cells (DCs). In peptide antigen presentation, major histocompatibility complex (MHC) class I molecules bind a short peptide (8-10 residues) in the endoplasmic reticulum to activate conventional CD8+ T cells, while MHC class II molecules bind a relatively longer peptide (~20 residues) in endocytic compartments to activate conventional CD4+ T cells1,4. In contrast, lipid-specific T cells are activated by CD1 proteins with lipid antigens loaded mainly in endocytic compartments5,6. Lipid antigen presentation requires the supply of lipid metabolites produced in lipid metabolism7,8,9,10 and the loading of functional lipid metabolites to CD1 proteins in endocytic compartments5,6. In this context, various cellular factors modulating lipid antigen presentation, especially in environmental exposure of lipophilic pollutants and immune disorders, are critical to be defined. In this study, we used transcriptomic analysis, image profiling, and cellular and populational imaging analysis of human monocyte-derived DCs to determine the endocytic protein trafficking contributing to lipid antigen presentation in pollutant exposure. Certainly, this combined platform can be applied to studying subcellular protein trafficking and colocalization in different biological processes.
Technically, subcellular protein localization was usually demonstrated using confocal microscopy and statistically analyzed in a limited number of detected cells1,2,3,11. Moreover, flow cytometry has been broadly applied to gate cell populations with co-stained signals of multiple proteins at a cellular level12; however, this lacks a detailed visualization of subcellular protein colocalization. To achieve comprehensive and statistical analyses of percentage protein colocalization at both cellular and population levels, we incorporate imaging profiling and analysis approaches to determine the features of protein colocalization with biological relevance. Specifically, we use imaging flow cytometry to detect the colocalization of CD1d and lysosomal-associated membrane protein 1 (Lamp1) proteins in this study. Quantitative analysis of colocalized molecules was previously difficult to be performed at a populational scale. In this study, we adapted the ImageJ-Fiji program to examine the percentage of protein colocalization with a large number of co-stained cells at both populational and individual cellular levels. Specifically, we measured co-localized areas, intensity, and populational size to support the conclusion that the CD1d protein was largely retained in late endocytic compartments of human DCs in exposure to a lipophilic pollutant benzo[a]pyrene (BaP)13. This combined cellular and population imaging analysis provided highly reproducible, comprehensive, and statistically significant results of CD1d-Lamp1 colocalization relevant to inhibited lipid antigen presentation.
The transcriptome of BaP-exposed human DCs strongly supported the hypothesis that endocytic lipid metabolism and CD1d endocytic trafficking were impaired in BaP exposure. To test this hypothesis, we applied imaging flow cytometry to profile the images of DC population that were co-stained with multiple proteins including CD1d, endocytic markers, and DC markers. Finally, co-stained cells were statistically analyzed to demonstrate the percentage of intensity and areas of CD1d and Lamp1 colocalization.

<table><tbody><tr><td>Transcriptomics</td><td>Illumina</td><td>HiSeq 2500 v4</td><td>Illumina HiSeq system</td></tr><tr><td>ImagingStream X</td><td>Millipore</td><td>100220</td><td>Imaging flow cytometry</td></tr><tr><td>mirVana miRNA Isolation Kit</td><td>Thermo Fisher</td><td></td><td>Total RNA extraction</td></tr><tr><td>Agilent RNA 6000 Pico Kit</td><td>Agilent</td><td></td><td>Total RNA QC analysis</td></tr><tr><td>Veriti 96-Well Fast Thermal Cycler</td><td>Thermo Fisher</td><td></td><td>PCR, enzyme reaction</td></tr><tr><td>NEBNext Poly(A) mRNA Magnetic Isolation Module&amp;nbsp;</td><td>New England Biolab</td><td></td><td>PolyA RNA purification</td></tr><tr><td>Automated SMARTer Apollo system</td><td>Takara</td><td></td><td>PolyA RNA purification</td></tr><tr><td>NEBNext Ultra RNA Library Prep Kit for Illumina</td><td>New England Biolab</td><td></td><td>Library preparation</td></tr><tr><td>Agencourt AMPure XP magnetic beads</td><td>Beckman Coulter</td><td></td><td>DNA purification&amp;nbsp;</td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent</td><td></td><td>Library QC, size distribution analysis</td></tr><tr><td>Agilent High Sensitivity DNA Kit</td><td>Agilent</td><td></td><td>Library QC, size distribution analysis</td></tr><tr><td>QuantStudio 5 Real-Time PCR System (Thermo Fisher)</td><td>Thermo Fisher</td><td></td><td>Library quantification</td></tr><tr><td>NEBNext Library Quant Kit</td><td>New England Biolab</td><td></td><td>Library quantification</td></tr><tr><td>cBot</td><td>Illumina</td><td></td><td>Library cluster generation</td></tr><tr><td>TruSeq SR Cluster Kit v3 - cBot &amp;ndash; HS</td><td>Illumina</td><td></td><td>Library cluster generation</td></tr><tr><td>HiSeq 1000</td><td>Illumina</td><td></td><td>Sequencing</td></tr><tr><td>TruSeq SBS Kit v3 - HS (50-cycles)</td><td>Illumina</td><td></td><td>Sequencing</td></tr><tr><td>Phycoerythrin/Cyanine 7 (PE/Cy7)</td><td>Bio Legend</td><td>L243</td><td></td></tr><tr><td>Phycoerythrin/Cyanine 5 (PE/Cy5)</td><td>Bio Legend</td><td>3.9</td><td></td></tr><tr><td>Brilliant violet 421</td><td>Bio Legend</td><td>L161</td><td></td></tr><tr><td>PE-anti-mouse IgG2b</td><td>Bio Legend</td><td>51.1</td><td></td></tr><tr><td>Alexa Fluor 647 (AF647)</td><td>Bio Legend</td><td>CD107a(H4A3)</td><td></td></tr></tbody></table>

integrate imaging flow cytometry and transcriptomic profiling to evaluate altered endocytic cd1d trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

The lipophilic pollutant BaP alters endocytic gene clusters in human DCs. Human monocyte-derived DCs from each donor (n=3) were incubated with BaP and sorted for&#160;conventional DCs, which were further used for RNA extraction and transcriptomic analysis as described. Upon the normalization of gene expression, altered genes between BaP-exposed and non-exposed groups were clustered according to the functional correlation of differentially expressed genes. We input the altered gene list to...

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Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...

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Research

JoVE Journal

Immunology and Infection

6.6K Views.  University of Cincinnati College of Medicine. Deze methoden integreren de volgende generatie sequencing en flow cytometrie beeldvorming analyse. Het kan helpen bij het beantwoorden van interessante vragen, zoals de vraag of sommige fenotypische veranderingen zijn implicate, in plaats van de transcriptoom, vooral in de blootstelling van verontreinigende stoffen omgeving. Het belangrijkste voordeel van deze eenvoudige methode voor ons is dat we het kunnen gebruiken om de colocalisatie van biologisch belangrijke eiwitten te meten, om de res...

Video: Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Biology

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Bioengineering

Screening of β2 Integrin Activation Inhibitors in Human Neutrophils

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

Author Spotlight: Development of a Method for Identifying Small Molecular Antagonists of &#946;2 Integrin Activation 

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

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