This work is supported by the National Natural Science Foundation of China General Program (31370878), State Key Program (31630023) and Innovative Research Group Program (81621002).

<ol>
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The authors have nothing to disclose.

AFM-based single-cell force spectroscopy has evolved to be a powerful tool to address the biophysical properties of living cells. For those applications, the cantilever needs to be functionalized properly in order to probe specific interactions or properties on the cells of interest. Here, the methods for coupling single T cell and single micron-sized bead to the tip-less cantilever are described respectively. To attach a single T cell to the cantilever, a biocompatible glue was chosen as cell adhesive. It is a specially...

Atomic force microscopy (AFM), a versatile tool, has found many applications in cell biology research1,2,3,4,5. Apart from its high-resolution imaging capability, the native force-probing feature allows biophysical properties of living cells to be investigated directly in situ at the single-cell level6,7. These include the rigidities of subcellular structures or even whole cells8,9,10,11,12, specific ligand/receptor binding strengths at the single-molecule level on the cell surface13, and adhesion forces between single-pairs of solid particles and cells or between two cells1,2,14,15. The latter two are often categorized as single-cell force spectroscopy (SCFS)16. Owing to the readily available cantilevers with various spring constant, the force range accessible to AFM is rather broad from a few piconewtons (pN) to micronewtons (&#181;N), which adequately covers the entire range of cellular events involving forces from a few tens of pN, such as receptor-based single-molecule binding, to nN, such as phagocytic cellular events15. This large dynamic force range makes AFM advantageous over other force-probing techniques such as optical/magnetic tweezers and a biomembrane force probe, as they are more suitable for weak-force measurements, with force typically less than 200 pN17,18. In addition, AFM can function as a high-precision manipulator to deliver various stimuli onto single cells in a spatiotemporally defined manner4,19. This is desirable for the real-time single-cell activation studies. Combined with live-cell fluorescence imaging, the subsequent cellular response to the specific stimulus can be monitored concurrently, thus making AFM-based SCFS exceedingly robust as optical imaging providing a practical tool to probe cellular signaling. For instance, AFM was used to determine the strains required to elicit calcium transients in osteoblasts20. In this work, calcium transients were tracked fluorescently through calcium ratiometric imaging after the application of localized forces on cultured osteoblasts with an AFM tip. Recently, AFM was employed to stretching collagen fibrils on which hepatic stellate cells (HSC) were grown and this mechano-transduced HSC activation was real-time monitored by a fluorescent Src biosensor, whose phosphorylation as represented by the fluorescence intensity of the biosensor is correlated with HSC activation3.
In AFM-based SCFS experiments, proper functionalization of AFM cantilevers is a key step toward successful measurements. Since our research interest focuses on immune cells activation, we routinely functionalize cantilevers with particulate matters such as single solid particles that can trigger phagocytosis and/or strong immune responses4,14,15 and single T cells that can form an immune synapse with antigen presenting cells, such as activated dendritic cells (DC)2. Single solid particles are normally coupled to a cantilever via an epoxy glue in the air environment, whereas single T cells, due to their non-adhesive nature, are functionalized to a cantilever via a biocompatible glue in solution. Here, we describe the methods to perform these two types of cantilever modification and give two associated applications as well. The first application is to probe T cell/DC interactions with AFM-SCFS to understand the suppressive mechanism of regulatory T cells from the cell mechanics point of view. The second one involves combining AFM with live-cell fluorescence imaging to monitor the cellular response of macrophage to a solid particle in real-time to reveal the molecular mechanism of receptor-independent phosphatidylinositol 4,5-bisphosphate (PIP2)-Moesin mediated phagocytosis. The aim of this protocol is to provide a reference framework for interested researchers to design and implement their own experimental settings with AFM-based single-cell analysis for immunological research.

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;l pipette tip</td>
			<td>Thermo Fisher</td>
			<td>104-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm diameter culture dish</td>
			<td>NALGENE nunc</td>
			<td>150462</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>JET</td>
			<td>TCP011006</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM Cantilever</td>
			<td>NanoWorld</td>
			<td>Arrow-TL1-50</td>
			<td>tipless cantilever</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>7604</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocompatible glue</td>
			<td>BD Cell-Tak</td>
			<td>354240</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation Cocktail</td>
			<td>STEMCELL</td>
			<td>19852C.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DC2.4 cell line</td>
			<td>A gift from K. Rock (University of Massachusetts Medical School, Worcester, MA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-coated magnetic particles</td>
			<td>STEMCELL</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>GENEray</td>
			<td>Generay-E1101-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>ERGO</td>
			<td>7100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>twbio</td>
			<td>00019</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Ex Cell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocker</td>
			<td>STEMCELL</td>
			<td>18731</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>local vender (Hai Men Lian Sheng)</td>
			<td>HX-E37</td>
			<td>24mm diameter, 0.17mm thinckness</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>JinTong department of laboratory and equipment management, Haimen&#160;</td>
			<td>N/A</td>
			<td>customized</td>
		</tr>
		<tr>
			<td>H2O2 (30%)</td>
			<td>Sino pharm</td>
			<td>10011218</td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sino pharm</td>
			<td>80120892</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>51558</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>STEMCELL</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh nylon strainer</td>
			<td>BD Falcon</td>
			<td>REF 352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Moesin-EGFP</td>
			<td>N/A</td>
			<td></td>
			<td>cloned in laboratory</td>
		</tr>
		<tr>
			<td>Mouse CD25 Treg cell positive isolation kit</td>
			<td>STEMCELL</td>
			<td>18782</td>
			<td>Component: FcR Blocker,Regulatory T cell Positive Selection Cocktail, PE Selection Cocktail, Dextran RapidSpheres,</td>
		</tr>
		<tr>
			<td>Mouse CD4+ Tcell isolation kit</td>
			<td>STEMCELL</td>
			<td>19852</td>
			<td>Component:CD4+T cell isolation Cocktail, Streptavidin RapidSpheres, Rat Serum</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Lanyi chemical products co., LTD&#65292; Beijing</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1022-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PE selection cocktail</td>
			<td>STEMCELL</td>
			<td>18151</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>PLC&#948;-PH-mCherry</td>
			<td>Addgene</td>
			<td>36075</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres 6.0&#956;m</td>
			<td>Polysciences</td>
			<td>07312-5</td>
			<td></td>
		</tr>
		<tr>
			<td>polystyrene round bottom tube</td>
			<td>BD Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>STEMCELL</td>
			<td>13551</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7</td>
			<td>&#160;ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-2</td>
			<td>Peprotech</td>
			<td>Peprotech, 200-02-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Regulatory T cell positive selection cocktail</td>
			<td>STEMCELL</td>
			<td>18782C</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-coated magnetic particles</td>
			<td>STEMCELL</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection kit</td>
			<td>Clontech</td>
			<td>631318</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% EDTA</td>
			<td>Life</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>JD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x objective NA 0.8</td>
			<td>Zeiss</td>
			<td>420650-9901</td>
			<td>Plan-Apochromat</td>
		</tr>
		<tr>
			<td>Atomic force microscope</td>
			<td>JPK</td>
			<td>cellHesion200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman coulter</td>
			<td>Allegra X-12R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence imaging</td>
			<td>home-made objective-type total internal reflection fluorescence microscop based on a Zeiss microscope stand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>HERACELL 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Zeiss</td>
			<td>Observer A1 manual</td>
			<td></td>
		</tr>
	</tbody>
</table>

functionalization of atomic force microscope cantilevers with single-t cells or single-particle for immunological single-cell force spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

Figure 4A shows typical force-distance curves from the binding interaction between single-T cell and single-DC in one approach-retract cycle. The light red curve is the extension curve and the dark red one is the retraction curve. Since the extension curve is typically used for indentation or rigidity-analysis, here only the retraction curve is concerned for cell adhesion. The minimum value (the green circle) in the curve gives a measure of the maximum adhesi...

Watch this Scientific Journal Video about Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy at JoVE.com

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

functionalization-atomic-force-microscope-cantilevers-with-single-t

Research

JoVE Journal

Immunology and Infection

7.3K Views.  Peking Union Medical College. We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Video: Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the flexibility of a VA approach in the analysis of biological datasets. Examples of these datasets in immunology include flow cytometry, Luminex data, and genotyping (e.g., single nucleotide polymorphism) data. Contrary to the traditional information visualization approach, VA restores the analysis power in the hands of analyst by allowing the analyst to engage in real-time data exploration process. We selected the VA software called Tableau after evaluating several VA tools. Two types of analysis tasks analysis within and between datasets were demonstrated in the video presentation using an approach called paired analysis. Paired analysis, as defined in VA, is an analysis approach in which a VA tool expert works side-by-side with a domain expert during the analysis. The domain expert is the one who understands the significance of the data, and asks the questions that the collected data might address. The tool expert then creates visualizations to help find patterns in the data that might answer these questions. The short lag-time between the hypothesis generation and the rapid visual display of the data is the main advantage of a VA approach.

Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...