The research is supported by National Institutes of Health grants AI-52381, CA138623 and Kentucky Lung Cancer Research Board and institutional support from James Graham Brown Cancer Center.

Methodology
Description of Microscope
Live cell imaging experiments performed using TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300. The microscope equipped with heating stage. A cool snap HQ digital B/W CCD (Roper Scientific) camera and LAMDA 10-2 optical filter changer (Sutter instrument company) is attached to microscope. Excitation and emission wavelengths are controlled with filter wheels and controlled by Lamba 10-2 filter wheel controller, Sutter Instruments Co. Exposure time 500 ms should be enough to view RFP or GFP in live cells. Hardware control and acquisition of images are controlled by Metamorph software. The choice of the filters for the present study are, filter sets S480/20x, S525/40m and S565/25x, S620/60m for GFP and RFP, respectively; EGFP/DsRed dual dichroic, Chroma Technology. This filter wheel can accommodate up to six filter sets. Microscope is attached with Prior Proscan stage controller with Joy stick. All these hardware attachments can be controlled by Metamorph software. The microscope is also attached with Micromanipulators to hold the micropipettes.
A. Measuring Leukotriene B4 Directed Dendritic Cell Migration
Using the above described microscope setting, we developed methods for following ligand directed dendritic cell migration in real time. Two micromanipulators (Narishige) are attached to the microscope. The chemotaxis of mouse bone marrow derived dendritic cells (BMDCs) towards LTB4 gradient was recorded. Here, we describe the methods to prepare the micro-pipettes using micro-pipette puller, loading the ligand into micro-pipette and setting up the chemotaxis experiment on the heated stage of the microscope to monitor directional migration of cells. This method can be applied to test the efficacy of distinct chemoattractants in inducing migration as well as effect of inhibitors of chemotaxis in live cells. The isolation of BMDCs21 from mouse has been described in several publications including JoVE22.
1. Preparation of BMDCs
<ol>
	<li>Plate the BMDCs (after culturing for 10 days) on to the cover slip bottom Fluoro dish and allow them to grow for 16 hr ahead of the experiment.</li>
	<li>Wash the cells with 3 mL of 1X PBS at least 2 times (by adding 3 mL of 1X PBS to the plate and aspirating the buffer out).</li>
	<li>Add 2 mL of 1XPBS to plate. The cells are now ready for the experiment. Keep the cells in 37 &deg;C incubator prior to experiment.</li>
 </ol>
Preparation of ligand loaded micro pipette:
<ol start="4">
	<li>Fix the glass capillary tubes (Glass standard, 0.75 mm x 0.4 mm; 6", Cat# 625500, A.M. Systems, INC) on the vertical micropipette puller (Narishige International USA, Inc).</li>
	<li>Keep the temperature at 52 &deg;C and allow the glass to melt and pull apart to form sharp edge tips. </li>
	<li>Fix the pipette onto the micro-pipette holder.</li>
	<li>Smooth the rough edges of micro pipette using Micro forge M-900 (Narishige International USA, Inc) by watching under microscope. </li>
	<li>Prepare at least 10-20 pipettes for each experiment. 
Note: The micropipettes can also be purchased from World Precision Instruments (&mu; TIP, T1801TW1F).</li>
	<li>Take desired concentration (500 &mu;L of 100 nM LTB4 in the current experiment) of ligand into 1 mL effendorf tube and keep the micro pipette tip in the solution and allow the solution to enter into the micro pipette (back filling). </li>
	<li>Take 1 mL of ligand and into the syringe (1 cc tuberculin syringe) and slowly fill the ligand using Micro-Fill needle (MF 28G-5, World Precision Instruments) into micro pipette from top. </li>
	<li>Attach the tubing, which fits tightly to the pipette to pipette and tube to needle (21 G 11/2) attached to 1 mL syringe, which is filled with ligand.</li>
	<li>Now, carefully move the ligand filled micro-pipette to the microscope stage and fix onto micro manipulators. </li>
	<li>Apply little pressure from syringe to make sure ligand is slowly releasing from the tip.</li>
	<li>Bring the plated immature BMDCs to the microscope and keep them on the heated plate (37 &deg;C) stage.</li>
	<li>Remove the lid of fluoro dish.</li>
	<li>Focus the cells using oil immersion 60X objective lens. </li>
	<li>Carefully, bring down the ligand loaded pipette using 'Coarse Manual Manipulator MN-188NE' to the proximity of the cells.</li>
	<li>Focus the pipette using 'Hydraulic Fine Micromanipulator MN-188 NE'.</li>
	<li>Apply little pressure from syringe to make sure that ligand is releasing into dish. </li>
	<li>Set the image acquisition parameters to acquire in bright filed (10 ms exposure) in every 15 sec for 2 hrs using Metamorph software.</li>
	<li>Keep monitoring every 30 min for progression of migration towards ligand.</li>
	<li>If cells are crowded at pipette tip, change the location of the tip in the plate to continue the migration of experiment.</li>
</ol>
B. Measuring GPCR (BLT1) and Cytosolic Protein (&beta;-arrestin) Interactions, Translocation in Live Cells
1. Preparation of Plasmid DNA Constructs
The details of DNA plasmid constructs were described in Jala et al. (2005)18.
2. Transfection of Human BLT1-RFP and &beta;-Arrestin-GFP into RBL-2H3 Cells
<ol>
<li>Maintain the Rat Basophilic Leukomia (RBL-2H3) Cells (60 to 70% confluence) at 37 &deg;C in a humidified atmosphere of 95% air, 5% CO2 as monolayer cultures in growth media (DMEM supplemented with 15% FBS, 2 mM L-glutamine, 100 U/ mL penicillin and 100 &mu;g/ mL streptomycin) in T75 flasks.</li>
<li>Detach the cells from the dish/flasks by using 6 mL of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA) and incubating for 5 min at 37 &deg;C in a humidified atmosphere of 95% air, 5% CO2. Gently shake the flasks to monitor the extent of detachment of the cells.</li>
<li>Add 6 mL of growth media to flask containing 6 mL of Trypsin-EDTA and collect the cells by mixing them by pipetting up and down multiple times. Transfer the cells into 15 mL falcon tubes.</li>
<li>Count the cells using a hemocytometer and take 4 x 106 cells in 15 mL centrifuge tubes and centrifuge at 480g rpm for 3 min and resuspend in 200 &mu;L of transfection media (DMEM, 20% FBS, 50 mM HEPES). If you plan to perform 3 transfections, prepare the cells and tranfection media accordingly by increasing cell number and the volume of transfection medium.</li>
<li>Prepare the plasmids to be transfected at the concentrations of at least 1.5 &mu;g/&mu;L. We prepare the DNA plasmid by using QIAGEN Maxi DNA plasmid kit. Add the individual or both plasmid DNAs encoding for human BLT1-RFP (25 &mu;g) and &beta;-arrestin1-GFP (15 &mu;g) to sterile electrophoresis cuvettes, (Bio-Rad # 16552088), which has 0.4 cm electrode gap.</li>
<li>Take 200 &mu;L of the above resuspended cells and place them in cuvettes containing either hBLT1-RFP or &beta;-arrestin1-GFP or both together and mix them gently with 1 mL sterile pipette.</li>
<li>Leave the above cuvettes at room temperature for 10 min.</li>
<li>Electroporate the cells in Gene Pulser II with voltage 250 v and capacity 500 &mu;F.</li>
<li>After electroporation let the cells stay at room temperature for 10 min.</li>
<li>Take 1 mL of regular growth medium and mix it with electroporated cells in the cuvettes.</li>
<li>Distribute 300 &mu;L of this mixture into 35 mm tissue glass bottom culture dishes (Fluoro dish, World Precision Instruments) containing 2 mL of regular growth media and incubate for 1 hr at 37 &deg;C in a humidified atmosphere of 95% air, 5% CO2. Allow the cells to adhere to bottom of the dish.</li>
<li>Replace the media after 1 h of incubation with regular growth media and allow them grow at 37 &deg;C in a humidified atmosphere of 95% air, 5% CO2 incubator for 18-24 hrs.</li>
</ol>
3. Live Cell Imaging
Acquisition of images
<ol>
<li>After 18-24 hrs, wash the cells 2 or 3 times with 2 mL of warm phenol red free RPMI containing 10 mM HEPES, pH 7.55 (Add 2 mL of warm media directly to plate and aspirate media. Repeat 2 or 3 times). Add 1.8 mL of warm phenol red free RPMI containing 10 mM HEPES.</li>
<li>Place 30 mm glass coverslip-bottomed culture dishes containing RBL-2H3 cells, transfected with hBLT1-RFP and &beta;-arrestin1-GFP on the heated microscope stage (37 &deg;C).</li>
<li>Collect the cell images at 600x magnification using 60 X objective Nikon Plan Apo 60X/1.4 numerical aperture oil immersion lens (available in our microscope system).</li>
<li>Put one oil drop on the 60 x objective lense and focus the cells with regular bright field transmitted light by touching the bottom of the plate.</li>
<li>To monitor the fluorescence of transfected proteins, switch from bright field (transmitted light) to fluorescence filter (RFP filter, if you want to see receptor or GFP, if you want to see &beta;-arrestin1).</li>
<li>Choose the bright and healthy cell, which expresses hBLT1-RFP on the cell surface using RFP filter and capture the image.</li>
<li>Then shift to GFP filter and make sure that &beta;-arrestin-GFP expressed in the cytoplasm and capture the image. Then pseudo color both the images, before capturing time lapsed live cell images to make sure that bleed through of fluorescence is not taking occurring between RFP and GFP.</li>
<li>Once the choice of your cell made, set the parameters according to your purpose using software (Metamorph Software in this instance).</li>
<li>In the current demonstration, sixteen bit images are acquired with the camera binning set to 1 X 1 combined with 60 X objective Nikon Plan Apo 60X/1.4 numerical aperture oil immersion lens.</li>
<li>Collect the fluorescence images for RFP and GFP fluorochromes simultaneously at 30 sec time interval (if the translocation/interactions are too fast, one can reduce the time intervals) of these using filter wheels controlled by Metamorph software.</li>
<li>The Camera exposure times set to 1000 msec for RFP and GFP. (Set the specific exposure times according to the fluorescent intensity of RFP and GFP).</li>
<li>First collect the images for 1 min without adding ligand, which serves as 0 time point.</li>
<li>Add the 200 &mu;L of ligand (from stock of 10 &mu;M to a final concentration of 1 &mu;M) after 1 min without disturbing the plate/or cell position.</li>
<li>The fluorescence images are collected for 60 min after adding the ligand and stored as TIFF images with increasing order of the file names.</li>
<li>All these files can be made as stack files with individual/combined fluorescence images with time stamps using the software.</li>
</ol>
Process of images and quantification of fluorescence of proteins and localization
<ol>
<li>Acquire the images with plain media (no cells) with RFP and GFP fluorescent filters and save them as background images. Use these images to subtract from actual data images obtained above.</li>
<li>Prepare the stack of images individually for RFP and GFP images.</li>
<li>Select/define the various regions (surface vs cytosolic) to measure the fluorescence intensity of RFP and GFP (BLT1 and &beta;-arrestin translocation)</li>
<li>Plot the amount fluorescence intensities as the function of time. This graph will provide the information about kinetics of translocation of given molecule.</li>
</ol>
Representative Results
A. Measuring Leukotriene B4 Directed Dendritic Cell Migration
The method described in this protocol was used to determine the dendritic cell migration towards leukotriene B4 (Figure 1 and attached video 1)21. We can apply similar methodology to determine the chemotactic capacity of cells to a particular ligand in live cells.
<img src="/files/ftp_upload/2315/2315fig1.jpg" alt="Figure 1" /> 
Figure 1. Mouse bone marrow derived dendritic cell migration towards 100 nM LTB4.
Video 1. Live cell migration of BMDCs towards LTB4. <a href="https://www.jove.com/files/ftp_upload/2315/Jala and Boddulrui_Video1.AVI">Click here to see video</a>.
B. Measuring GPCR (BLT1) and Cytosolic Protein (&beta;-arrestin) Interactions, Translocation in Live Cells
Upon completion of this procedure, one can obtain the following information in GPCR signaling events.
<ol class="lroman">
<li>The localization of GPCR and cytosolic proteins and nucleus (Figure 2).</li>
<li>Ligand induced interaction of surface GPCR (BLT1 in this case) with cytosolic protein (&beta;-arrestin in this case) (Fig 3).</li>
<li>Kinetics of receptor internalization and &beta;-arrestin translocation to membrane followed by internalization along with receptors (Figure 4) (Jala et al. 2005)18.</li>
<li>One can determine the ligand dependent receptor activation status in live cells (attached video 2) and determine critical motifs or processes involved in the receptor activation (Jala et al. 2005)18.</li>
</ol>
<img src="/files/ftp_upload/2315/2315fig2.jpg" alt="Figure 2" /> 
Figure 2. Expression of BLT1-RFP (A), &beta;-arrestin-GFP (B), pNuc-CFP (C) into RBL-2H3 cells. Color combined image shown in panel D.
<img src="/files/ftp_upload/2315/2315fig3.jpg" alt="Figure 3" /> 
Figure 3. Ligand induced translocation of receptor and &beta;-arrestin. Line scan of fluorescent intensities in the cells are shown.
<img src="/files/ftp_upload/2315/2315fig4.jpg" alt="Figure 4" /> 
Figure 4. Kinetics of receptor internalization and &beta;-arrestin translocation upon addition of 1 &mu;M LTB4. The fluorescence intensities were measured as a function of time at membrane and cytosolic locations of the cell.
Video 2 Live imaging of cells expressing BLT1-RFP and &beta;-arrestin-GFP upon the addition of 1 &mu;M LTB4. <a href="https://www.jove.com/files/ftp_upload/2315/Jala and Bodduluri_Video2.AVI">Click here to watch video</a>

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No conflicts of interest declared.

Live cell imaging is a powerful tool to demonstrate the function and interactions of specific proteins as they occur in real-time. The methods described in this manuscript clearly show that LTB4 can induce rapid migration of dendritic cells. These methods not only expand the aspects of LTB4 function to diverse cell types, they allow similar methods to be applied to a variety of other chemokines and testing their efficacy as chemotactic agents on different leukocyte sub populations. Fluorescence ima...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell lines:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells.</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CRL-2256</td>
<td>&nbsp;</td>
<tr>
<td>Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Delbecco's modified Eagle&#x2019;s Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red free RPMI or DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11835-030</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin (10000 U/mL)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>HEPES (1M)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Others:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish)</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>FD35-100</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>16552088</td>
<td>&nbsp;</td>
<tr>
<td>Instruments/software:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser II electroporater </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph Software</td>
<td>&nbsp;</td>
<td>Universal Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vertical Micro-pipette puller</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Forge M-900</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hadraulic Micromanipulator MO-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coarse Manual Manipulator, MN-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA constructs:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP)</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> cDNA of cytosolic protein tagged with GFP (&beta;-arrestin1-GFP in present study).</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

real-time imaging of leukotriene b4 mediated cell migration and blt1 interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Watch this Scientific Journal Video about Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with Î²-arrestin at JoVE.com

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

real-time-imaging-leukotriene-b4-mediated-cell-migration-blt1

Research

JoVE Journal

Immunology and Infection

12.7K 조회수.  University of Louisville. 본 논문은 특정 리간드에 leukocytes의 chemotactic 응답을 확인하고 라이브 셀 이미징 기술을 사용하여 세포 표면 수용체와 cytosolic 단백질 사이의 상호 작용을 식별하는 방법을 설명합니다.

비디오: Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...