This work was supported by University of Alberta Faculty of Medicine and Dentistry, The Friends of Garrett Cumming Research Chair Fund, HM Toupin Neurological Science Research Chair Fund, Muscular Dystrophy Canada, Canada Foundation for Innovation (CFI), Alberta Advanced Education and Technology (AET), Canadian Institutes of Health Research (CIHR), Jesse's Journey - The Foundation for Gene and Cell Therapy, the Women and Children's Health Research Institute (WCHRI), and Alberta Innovates Health Solutions (AIHS).
We would like to thank Dr. Steven Laval for supplying us with the full-length dysferlin plasmid. We would also like to thank Dr. Katsuya Miyake for technical advice.

Human fibroblast cells used in this study were used with approval from the Human Ethics Committee of the Faculty of Medicine and Dentistry at the University of Alberta.
1. Transfection of cells with full-length DYSF plasmid
<ol>
	<li>Culture fibroblast cells in a T225 flask containing 40 mL of growth media - 36 mL Dulbecco&#39;s Modified Eagle Medium (DMEM), 4 mL of fetal bovine serum (FBS), and 20 &#181;L of penicillin/streptomycin (P/S) - in a CO2 incubator at 37&#176;C. 
	NOTE: Cells should be cultured to approximately 70-80% confluency and should not be allowed to become completely confluent.</li>
	<li>To prepare cells for seeding, rinse the cells with 40 mL of phosphate buffered saline (PBS) twice, then add 5 mL of 0.05% trypsin to detach the cells. Return the cells to the 37 &#176;C CO2 incubator and incubate the cells for 5 min.
	<ol>
		<li>When the cells are fully detached, add 40 mL of growth media to stop the trypsin reaction.</li>
		<li>Count the cells using a hemocytometer or another cell counting device.</li>
	</ol>
	</li>
	<li>Seed 2 mL of cells at 1x105 cells/mL into 35 mm collagen-coated glass-bottom dishes. Incubate the cells overnight in a CO2 incubator at 37 &#176;C. 
	NOTE: Cells should be approximately 50-60% confluent the next day.</li>
	<li>Remove the growth media and replace it with 2 mL of serum-deprived media (growth media without FBS).</li>
	<li>Transfect the cells with full-length dysferlin plasmid using lipotransfection.
	<ol>
		<li>Prepare 150 &#181;L of serum-deprived media in a 1.5 mL tube (one tube for each dish to be transfected).</li>
		<li>Add 3 &#181;L of transfection reagent to each 1.5 mL tube containing serum-deprived media. Incubate it at least 5 min at room temperature.</li>
		<li>In a separate 1.5 mL tube, add 175 &#181;L of serum-deprived media and 3.5 &#181;g of plasmid DNA. 
		NOTE: Multiply the above amounts for each additional dish to be transfected.</li>
		<li>Combine 150 &#181;L of media containing plasmid DNA into a tube with 150 &#181;L of serum-deprived media and transfection reagent. Incubate it for 20 min at room temperature.</li>
		<li>Evenly distribute 300 &#181;L of media with plasmid and transfection reagent across the dish. Incubate the dishes for 24 h in a CO2 incubator at 37&#176;C.</li>
	</ol>
	</li>
	<li>Replace the media with fresh growth media containing serum and incubate it for an additional 24 h in a CO2 incubator at 37 &#176;C.</li>
</ol>
2. Preparation of cells for two-photon wounding assay
<ol>
	<li>Remove the media by pipetting and rinse the cells once with 1 mL of Tyrode&#39;s solution, then add 1 mL of fresh Tyrode&#39;s solution containing 1 &#181;L of 2.5 mM FM 4-64 dye. 
	NOTE: The final concentration of FM 4-64 dye is 2.5 &#181;M.</li>
	<li>If assessment of membrane repair in a calcium-depleted environment is desired, rinse cells with 1 mL of PBS and then add 1 mL of PBS containing 1 mM EGTA. 
	NOTE: As PBS will cause the cells to detach over time, the wounding assay should be completed as quickly as possible.</li>
</ol>
3. Two-photon laser wounding assay
<ol>
	<li>Using an inverted confocal microscope and associated program software, create a 0.2 &#181;m x 2 &#181;m target and place it at the edge of the cell membrane so that the target line overlaps and lies perpendicular to the direction of the cell membrane (Figure 2) .</li>
	<li>Use a 543-nm HeNe laser to excite FM dye signal and set the detection range for 600-760 nm. Use a 488-nm Argon laser to excite GFP signal (Figure 4) and set the detection range for 500-550 nm.
	<ol>
		<li>Create a 5 min time series of sequential image scans, imaging cells every 5 s.</li>
		<li>To generate a membrane lesion, bleach cells using a two-photon laser set to 820-nm, using 15% laser power with 10 iterations and bleach cells 25 s after the beginning of the time series.</li>
	</ol>
	</li>
	<li>To quantify FM 4-64 dye fluorescence, use the software to draw a 6 &#181;m x 6 &#181;m region of interest (ROI) and place it adjacent to the wounding location and within the cell (Figure 3). 
	NOTE: Avoid sampling areas of pixel oversaturation by using the &#34;Range Indicator&#34; function in the software - in this application, red pixels represent oversaturated pixels. Export raw fluorescence values into an electronic spreadsheet for statistical analysis.</li>
	<li>Calculate relative fluorescence values for each time point by subtracting the background value (mean fluorescence value of timepoints prior to membrane wounding) and dividing the net increase by the fluorescence value at t = 0 (Figure 5, 6).</li>
</ol>

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The authors have nothing to disclose.

Two-photon laser wounding of the cell membrane is a precise and versatile technique for assessing the dynamics of membrane resealing in vitro. In this article, we described a protocol for determining cell membrane resealing ability in dysferlinopathy patient cells using the two-photon laser wounding assay. Our results show that dysferlinopathy patient cells are defective in cell membrane resealing, which is consistent with findings by other researchers and which further highlights the striking similarities in cell membrane resealing dynamics between muscle and non-muscle cell types3,11,15,22. We also observed that patient cells transfected with a plasmid containing the full-length dysferlin sequence were rescued from their defective membrane resealing phenotype, demonstrating that full-length dysferlin plasmid can rescue phenotype in non-muscle cell types as well as in muscle27. 
Together, our results reinforce the feasibility and reliability of the two-photon membrane wounding assay as a gold-standard in measuring cell membrane resealing ability in vitro. However, the cost and availability associated with two-photon microscopy could be a potential limiting factor for some research groups seeking to utilize the two-photon membrane wounding assay. Furthermore, our setup utilizes an inverted confocal microscope, which is well-suited to in vitro work but may be less appropriate for in vivo imaging.
It is important to note that the precision of the two-photon laser alignment is critical to the success of the technique. In our experience, troubleshooting of laser alignment was the most common issue encountered. Other issues which could affect the ability to score successful hits during laser wounding include the levelling of the glass-bottom dish on the stage, as well as the orientation of the cells in solution. 
The assay is applicable to current and future studies investigating disease states in which cell membrane integrity or resealing is perturbed, and the efficacy of therapies, such as gene therapy and antisense oligonucleotide-based therapy, which would benefit from having an effective assay for characterizing membrane dynamics so that novel therapeutic options can be explored28,29.

The cell membrane of eukaryotic cells consists of a double-layer of protein-studded phospholipids which defines the intra/extracellular environment of the cell and is essential for maintaining cellular homeostasis and cell survival. Cell membrane injuries arising from mechanical or chemical insults are commonplace in various mammalian cell types, including skeletal and cardiac muscle, stomach, and lung cells1,2,3,4. In addition to injuries consequent to daily physiological function, cell membranes can also be damaged by environmental insults, bacterial toxins, and ischemic reperfusion5. Failure to reseal ruptures in the cell membrane leads to an unregulated influx of extracellular Ca2+- along with other potentially toxic extracellular components - into the cell, triggering downstream signal cascades which can quickly result in cell death1,4,5,6.
To date, there have been several proposed models for membrane repair in cells. Different repair mechanisms may be activated depending on the size and nature of the membrane rupture. For example, it is suggested that cell membranes may utilize lateral flow or protein clogging to repair small disruptions (&#60;1 nm). The lateral fusion model proposes that membrane ruptures are rapidly mended through lateral recruitment of dysferlin-containing membrane7, while the protein clogging model suggests that small perforations are mended through protein aggregations (mostly annexins)8. Conversely, larger membrane lesions trigger Ca2+-dependent, dysferlin-mediated vesicle fusion and formation of a repair patch. In the repair patch model, a rapid Ca2+ influx into the cell triggers the recruitment of multiple proteins (dysferlin, annexins, mitsugumin-53, and EDH proteins) which form a complex or &#34;patch&#34;, along with a fusion of intracellular vesicles or lysosomes at the site of membrane damage4,9,10,11,12. It is important to note that these models are not necessarily mutually exclusive and may work in concert to facilitate membrane repair. Failure to properly reseal cell membrane damage is associated with multiple disease states, including muscular dystrophy (dysferlinopathy - including Miyoshi myopathy, limb-girdle muscular dystrophy type IIB, and distal myopathy with anterior tibial onset)13, cardiomyopathy14, and Chediak-Higashi syndrome (CHS)15.
Given that proper cell membrane integrity and resealing plays such an important role in health and disease, a deeper understanding of the underlying molecular mechanisms of cell membrane repair would be beneficial in the search for novel therapeutic strategies. It is, therefore, necessary to possess appropriate experimental techniques to&#160;monitor the kinetics and evaluate the ability of cell membrane repair. Several in vitro methods for modeling membrane repair have been designed. One strategy involves mechanical injury, which can be facilitated through cell scraping with a pipette/surgical blade/razor or by rolling glass beads over the cells16. However, this type of mechanical injury generates larger lesions, and creates a high degree of variation in cell injury, both within and between cultures.
Another method of generating membrane wounds is laser ablation by two-photon microscopy. In contrast to traditional laser confocal microscopy that employs single photon excitation, the two-photon laser simultaneously uses two long-wavelength, low-energy photons to facilitate the excitation of a high-energy electron17. This non-linear process results in excitation exclusively within the focal plane and not along the entire light path17 (Figure 1). This reduced excitation volume helps to minimize photodamage when imaging live cells18,19. Researchers are therefore able to generate precise lesions in the cell membrane and monitor membrane resealing in real time by using fluorescent dyes and observing changes in the fluorescence intensity as the cell membrane ruptures and then reseals.
This approach has been used repeatedly to study membrane wounding in vitro and in vivo and in multiple cell types20,21. For example, membrane repair defects in fibroblasts and myotubes derived from dysferlinopathy patients were assessed using this technique22,23. Also, single muscle fibers isolated from mice were used to monitor repair patch formation13,24,25. The movement of fluorescently tagged proteins can also be observed during membrane repair in single muscle fibers9. Furthermore, the process of sarcolemmal repair following two-photon laser wounding in zebrafish embryos can be observed in real-time in vivo26.
In this article, we outline a methodology for assessing cell membrane repair dynamics in fibroblasts using two-photon laser wounding, although this methodology can be applied to various cell types for the purpose of quantifying plasma membrane resealing ability in vitro. In this method, cells are incubated with FM4-64, a lipophilic, cell-impermeable dye which quickly fluoresces as it binds to negatively charged phospholipids within the cytoplasm upon entering the cell through the membrane lesion (Figure 2&#38;3). Quantification of dye fluorescence adjacent to the membrane lesion allows for monitoring the time that it takes for the cell membrane to reseal itself. To exemplify the utility of this method, we use dysferlinopathy patient fibroblasts transfected with GFP-conjugated full-length dysferlin (DYSF) plasmids to assess the rescue of cell membrane repair.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">11320033</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">35mm collagen-coated glass-bottom dishes</td>
			<td style="width:71px;">MatTek</td>
			<td style="width:119px;">P53GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Serum-deprived media</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transfection reagent</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15338100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">FM 4-64 Dye</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">T13320</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Tyrode&#8217;s Salts Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T2397</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Confocal laser scanning microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Chameleon Two-photon laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
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cell membrane repair assay using a two-photon laser microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Healthy human fibroblasts, non-treated dysferlinopathy patient fibroblasts, and patient fibroblasts transfected with a plasmid containing the full-length dysferlin sequence were subjected to two-photon laser wounding to assess membrane resealing ability in real time. Healthy human fibroblast cells displayed low levels of FM 4-64 fluorescence activation following laser wounding and non-treated patient fibroblasts exhibited a high degree of relative fluorescence intensity following injury (Figure 5&#38;6). Patient cells that were transfected with full-length dysferlin plasmid showed reduced relative FM 4-64 fluorescence intensity compared with non-treated patient controls and were comparable with fluorescence values observed in the healthy control (Figure 5&#38;6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56999/56999fig1.jpg" /> 
Figure 1. One-photon versus two-photon microscopy. In a traditional one-photon microscope system, a single high energy photon (UV or visible spectrum) is used to excite a fluorophore (A). In a two-photon system, two lower-energy photons (near infrared) are used to excite a fluorophore (B). Depicted within the cylinders is the fluorescence signal generated throughout the focal plane, demonstrating how the two-photon laser system can focus excitation light to within a specific region (B) and exclude light from out-of-focus planes (A). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56999/56999fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56999/56999fig2.jpg" /> 
Figure 2. Placing a target at the cell membrane. Using software, a 0.2 &#181;m x 2 &#181;m target is drawn and placed overlapping and perpendicular to the cell membrane.The arrow indicates the target. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56999/56999fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56999/56999fig3.jpg" /> 
Figure 3. Fluorescence signal from FM dye as it infiltrates the cell through membrane rupture. As the cell membrane ruptures, lipophilic FM 4-64 dye infiltrates the cell and fluoresces as it binds negatively-charged phospholipids within the cytoplasm. Using software, a region of interest (ROI) can be drawn (white box) and fluorescence values within the box can be used to calculate relative fluorescence changes within the cell. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56999/56999fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56999/56999fig4.jpg" /> 
Figure 4. Fluorescence signal from GFP-conjugated full-length DYSF plasmid. Following plasmid transfection, patient fibroblast cells express full-length DYSF protein. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56999/56999fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56999/56999fig5.jpg" /> 
Figure 5. Quantification of fluorescence over time. Fluorescence values calculated by computer software from within the ROI can be used to determine relative fluorescence changes over time for each treatment group. The arrow indicates the time of laser ablation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56999/56999fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56999/56999fig6.jpg" /> 
Figure 6. Change in relative fluorescence over time. Relative fluorescence (RF) values for each time point are calculated by subtracting the background fluorescence value (calculated as the mean of the fluorescence values from the timepoints prior to membrane wounding) from the given fluorescence values (Signal Intensity - Background = &#916;F) and dividing the net increase (&#916;F) by the fluorescence value at t = 0 (F0). Therefore, RF = &#916;F/F0. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56999/56999fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell Membrane Repair Assay Using a Two-photon Laser Microscope at JoVE.com

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

cell-membrane-repair-assay-using-a-two-photon-laser-microscope

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.3K Views.  University of Alberta Faculty of Medicine and Dentistry. Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Video: Cell Membrane Repair Assay Using a Two-photon Laser Microscope

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMR&alpha;) with high purity9-13. The recombinant sGMR&alpha; was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMR&alpha; than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMR&alpha; yielded the data with high plasticity and reliability. GM-CSF binding to sGMR&alpha; was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMR&alpha; is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

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, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...