The authors would like to acknowledge Dr. Wade Sigurdson for providing valuable input in the design of the flow apparatus.

1. Assemble the Flow Apparatus
<ol>
	<li>Configure the parts listed in the Table of Materials according to the schematic in Figure 1 with the considerations discussed below. 
	NOTE: For convenience, the flow apparatus is divided into two sides, the green side (everything upstream of the slide to the media flasks), and the orange side (everything downstream of the slide to the media flasks).
	<ol>
		<li>Ensure that all of the flow apparatus is air tight to prevent leaks, with the sole exception of the media flasks (Figure 1, 1). To accomplish this, apply plumbers tape to any male threading before assembling except for the pulsation dampener (PD) and 2 &#181;m filter bottle (FB), which do not require plumbers tape as the rubber gaskets keep them airtight.</li>
		<li>Apply ear clamps at every barbed fitting that is under positive pressure during normal operation (i.e., downstream of the pump).</li>
		<li>Use color coded lab tapes to label the valve locations with an A or G (for attachment and growth, respectively), the pump location, the slide connection locations, and the 0.2 &#181;m filter connection.</li>
		<li>Determine the length of tubing to be used based on the distance between the flow system and microscope, keeping in mind that all the flow apparatus downstream of the pump to the flasks (majority of the orange side) should be in the secondary containment. Add approximately 1 m of extra tubing upstream of the slide (and preferably the bubble trap) to place within the microscope incubation chamber, as this ensures that all media reaching the slide will be at the correct temperature.</li>
		<li>Place the bubble trap as close as reasonably possible to the slide, preferably inside the incubation chamber during an experiment (bubbles often form along the tubing wall); however, keep in mind that it must be connected to a vacuum to operate.</li>
		<li>Ensure that the tubing between the FB and the 0.2 &#181;m disposable filter is about 0.5 m long.</li>
		<li>Add an approximately 2 cm magnetic stir bar to each media flask.</li>
		<li>Obtain some form of tubing clamps to act as shut-off valves (hemostats can be used).</li>
		<li>For ease of use, keep the flow apparatus in an autoclavable basket. It can be helpful to have a second smaller basket in a larger one to allow easy separation of the green side and the orange side.</li>
		<li>For the attachment flask, using a 4 mm drill bit, drill an extra hole in the rubber stopper to accommodate the thermal probe (take care not to go through another hole). To get the tubing through the ports, push the tubing through with tweezers; once through, clamp the tubing to hold it in place, and then pull the tweezers back out. 
		NOTE: If it is not possible to add the extra hole to the rubber stopper, a wide-mouth screw bottle with a four-port screw cap may work in place of the flask and rubber stopper.</li>
	</ol>
	</li>
	<li>Once the flow system has been fully assembled, close the valves of both green and orange side growth flasks. Use water with the attachment flask tubing and a graduated cylinder to calibrate the peristaltic pump according to the manufacturer&#39;s instructions.</li>
</ol>
2. Perform an Experiment
<ol>
	<li>The day before the experiment, begin pre-heating the microscope incubation chamber to 37 &#176;C, and prepare an overnight culture of a fungal strain (fluorescence is not required).</li>
	<li>Gather single use components and pump, and place in a sterile biosafety cabinet.</li>
	<li>Remove the bubble trap and the temperature probe from the flow apparatus and place these in the biosafety cabinet.</li>
	<li>Detangle and organize the tubing, if necessary.</li>
	<li>Autoclave the flow apparatus, including the stir bars, for 30 min to ensure sterility; when finished, transfer to the biosafety cabinet.</li>
	<li>Attach the bubble trap, temperature probe, and all single use components (except the slide) as depicted in Figure 1.
	<ol>
		<li>For the 0.2 &#181;m filter (Figure 1, 11), remove the plunger from the 1 mL syringe to make it as an &#34;adapter&#34;. Force the tubing from the FB into this end, and attach the 0.2 &#181;m filter to the tubing leading to the growth flask.</li>
		<li>Apply silicone vacuum grease around the barb of the slide adapter (take care not to get any grease on the inside) prior to connecting it, as this helps prevent air leaks into the system.</li>
	</ol>
	</li>
	<li>Fill the attachment flask with 100 mL of 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose (YPD), and fill the growth flask with 200 mL of YPD. Ensure that the green side tubing reaches the media in each flask.</li>
	<li>Ensure that all valves are open. Attach the bubble trap to a vacuum, and connect the pump to the green side tubing downstream of the bubble trap.</li>
	<li>Pump the fluid at a flow rate of 3.3 mL/min to completely fill the green side, then dispense and discard approximately 1&#8211;2 mL of the media because the first couple of milliliters often contain dead cells or random debris. Ensure that the green side of the tubing is filled with media, and has no bubbles downstream of the bubble trap before proceeding.</li>
	<li>Fill the channel slide and the reservoir with YPD, taking care not to introduce bubbles.</li>
	<li>Connect the slide to the flow apparatus, and pump more fluid to create a buffer of about 0.5 m on the orange side. This is to prevent accidentally trapping air in the slide in the event of backflow.</li>
	<li>Prepare the flow apparatus for the transport to the microscope: Clamp closed the inlet and outlet of the bubble trap, and clamp the green and orange side attachment flask valves closed. Ensure that the screw caps for the PD and FB are tight as they can loosen during autoclaving.</li>
	<li>Disconnect the pump from the tubing to make transport easier. Then move all components, including the hotplate stirrer, into a secondary container near the microscope.</li>
	<li>Prepare the flow apparatus for imaging.
	<ol>
		<li>Attach the temperature probe to the hotplate stirrer and begin heating the attachment flask to 37 &#176;C. Stir the media at 300 rpm and maintain this for the whole experiment.</li>
		<li>Mount the slide on the microscope, and use tape if necessary to tightly secure it.</li>
		<li>Attach the bubble trap to a vacuum (do not undo the clamp yet).</li>
		<li>Connect the pump to the flow apparatus at the location indicated on Figure 1.</li>
		<li>Start the pump at a flow rate of 3.3 mL/min, allow it to run for approximately 5&#8211;10 s, and then remove the bubble trap inlet/outlet clamp.</li>
		<li>Allow the pump to continue running while the attachment flask heats up. Once media has circulated throughout the flow system, check for normal operation.
		<ol>
			<li>Check fittings for air leaking in upstream of the pump (some bubble formation is normal), or fluid leaking out downstream.</li>
			<li>Check that the growth media flask, PD, and FB are all dripping media from the inlet tube (if not, this could indicate a clogged filter, or an overtightened ear clamp).</li>
			<li>Using the microscope, check for attached or rolling cells on the channel slide. An excessive number of cells may indicate contamination during set-up, or that the polytetrafluoroethylene (PTFE) membrane of the bubble trap needs replacing.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Once the attachment flask and incubation chamber are both at 37 &#176;C, add enough overnight culture of the fungal cells to the attachment flask to reach 1 x 106 cells/mL. 
	NOTE: The volume to add in &#181;L can be calculated using this formula: 
	<img alt="Equation 1" src="/files/ftp_upload/58457/58457eq1.jpg" /></li>
	<li>Wait 15 min to allow the cells to acclimate.</li>
	<li>Open both green and orange side attachment flask valves while closing both growth flask valves to start the flow of cells.</li>
	<li>Wait for approximately 5 min to allow cells to reach the slide, and allow for initial focusing of the microscope (this time may need to be adjusted depending on the length of the green side tubing). During this time, adjust the microscope to the same imaging parameters used in previous experiments. If this is the first run, follow these steps:
	<ol>
		<li>Switch to a low magnification air objective.</li>
		<li>Find and focus on an attached cell or small budding cell.</li>
		<li>Configure condenser for K&#246;hler illumination, then switch to darkfield.</li>
		<li>Set the exposure time to 300 ms.</li>
		<li>Adjust the illuminating intensity until a small cell is dim yet clearly visible against the background (a signal to background ratio of approximately 7&#8211;8 for a budding daughter cell is a reasonable value). Note/mark the illuminating intensity for future experiments.</li>
		<li>Configure the software to acquire an image every 2 min over 2 h.</li>
	</ol>
	</li>
	<li>Begin image acquisition for the attachment phase. Check back after approximately 5 and 10 min to ensure that focus has been maintained. If not, attempt to adjust the focusing immediately after the next image is acquired.</li>
	<li>Immediately after the attachment phase has finished, save the file, and then open both green and orange side growth flask valves while closing both attachment flask valves. Take care not to bump the stage if any valves are inside the incubation chamber.</li>
	<li>Unplug the thermometer probe from the hotplate stirrer.</li>
	<li>Remove the attachment flask from the hotplate stirrer and place the growth flask in its place.</li>
	<li>Configure the software to acquire an image every 15 min over 22 h and begin image acquisition for the growth phase. Re-focusing should not be necessary, but it is highly recommended to check the flow apparatus after a few hours.
	<ol>
		<li>Check fittings for air leaking in upstream of the pump (again some bubble formation is normal), or fluid leaking out downstream</li>
		<li>Check that the growth media flask, PD, and FB are all dripping media (if not, this could indicate a clogged filter, an overtightened ear clamp, or a clog at a barbed fitting if the cells being used flocculate).</li>
		<li>Check the fluid level in the FB. If the media is approaching the top of the bottle (over 1.5 cm above the top of the filter), tighten both screwcaps (do not loosen them, as this flask is under pressure). If they will not tighten further, continue the experiment (though this may result in a leak), and replace the rubber gaskets on the PD and FB after the next cleaning.</li>
	</ol>
	</li>
	<li>When the growth phase acquisition has finished, save the file, and then open the green and orange side attachment flask valves which may make a noise as the pressure releases on the orange side. Pull up on the green side tubing coming from both media flasks until they are at least several centimeters above the media. Run the pump at a high speed (approximately 100 mL/min or hold the fast forward button on the pump) to remove all the media from the tubing, which makes cleaning much easier. When emptied, disconnect the flow apparatus from the pump, and remove it from the microscope.</li>
</ol>
3. Clean the Flow Apparatus
<ol>
	<li>Remove all non-autoclavable components (single use components, bubble trap, and temperature probe), and autoclave the flow apparatus for 30 min. Discard used single-use components, clean probe with 70% ethanol, and set aside bubble trap.</li>
	<li>After autoclaving is finished, discard media, and rinse and set aside media flasks. Then re-connect the bubble trap, connect an ibidi channel slide&#160;to be used for cleaning (reusable), and connect the flow system to the pump at the location shown in Figure 1.</li>
	<li>Clamp closed the orange side growth flask valve.</li>
	<li>Place approximately 200 mL of undiluted bleach into a beaker. Place the rubber stoppers into the bleach, and then start the pump at a high speed to circulate bleach throughout the flow apparatus (except all the filters). Once filled with bleach, stop the pump because leaving&#160;the pump on at a high speed can wear and break the tubing.</li>
	<li>After bleaching for 15 min, hold the rubber stoppers above the beaker and start the pump again to remove the bleach from the flow apparatus.</li>
	<li>Repeat steps 3.4 and 3.5 twice with excess water instead of bleach to rinse the flow system. During this time, clean the filters only with water because other cleaning agents will corrode or clog the filters.
	<ol>
		<li>Place the tubing that would normally connect to the 0.2 &#181;m media filter (coming from the 2 &#181;m FB) into the beaker water with the rubber stoppers from step 3.6.</li>
		<li>Disconnect the tubing attached to the inlet of the 20 &#181;m inline filter, which can usually be pulled apart with ease despite the ear clamp.</li>
		<li>Use a vacuum filter flask and a long section of tubing through a spare 3-hole stopper to create a vacuum system that can connect to the flow apparatus.</li>
		<li>Connect this vacuum system to the inlet of the 20 &#181;m filter inlet, and start the vacuum; this will pull water through the filters in the reverse direction, removing dead cells.</li>
		<li>Pull at least 200 mL of water through the filters, then remove the tubing from the water to empty the filter lines of water.</li>
		<li>Disconnect the vacuum system from the 20 &#181;m filter, and reconnect the filter to its normal tubing.</li>
	</ol>
	</li>
</ol>
4. Quantifying the Videos
NOTE: All files need to be converted to the tag image file (TIF) format to work. Additionally, to compare between experiments, it is critical that all images are taken with the same microscope and imaging parameters, as discussed above.
<ol>
	<li>Download and install ImageJ if not already installed.</li>
	<li>Download the supplemental macro file, and place it in the ImageJ\macros folder.</li>
	<li>Adjust the provided macro:
	<ol>
		<li>Open an image stack from a previous experiment in ImageJ, and select a time point with cells present.</li>
		<li>Select from the menu via &#34;Image | Type | 8-bit&#34;.</li>
		<li>Select from the menu via &#34;Image | Adjust | Threshold&#34;. Check the &#34;Dark Background&#34; box. Set the right side dropdown menu to Red.</li>
		<li>Adjust the lower value until all cells are covered in red with minimal excess noise (some non-cell speckling is okay and will be processed out by the macro). Make note of this lower value.</li>
		<li>Close both the Threshold window and the open image.</li>
		<li>Select from the menu via &#34;Plugins | Macros | Edit&#34;. When prompted to open a file: &#34;move up one folder level&#34;, then select the macros folder and open the flow biofilm quantification macro file.</li>
		<li>Change the 15 value in all instances of &#34;setThreshold(15, 255);&#34; to the value determined in step 4.3.4. Save the file and close this window.</li>
	</ol>
	</li>
	<li>Select from the menu via &#34;Plugins | Macros | Install&#34; and select the flow biofilm quantification file.</li>
	<li>Now, under the &#34;Plugins | Macros&#34; menu, six new options for various video quantifications appear. Run the Complete analysis and select the attachment and growth video files when prompted to perform all available analyses on the acquired data and automatically generate output files.</li>
</ol>

<ol>
	<li>Pankhurst, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidiasis+(oropharyngeal).">Candidiasis (oropharyngeal).</a> BMJ clinical evidence. 2012, 1304 (2012).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., L&#243;pez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Revista iberoamericana de micolog&#237;a. 18 (4), 163-170 (2001).</li><li>Nobile, C. J., Mitchell, A. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+biofilm:+a+fungal+perspective.">How to build a biofilm: a fungal perspective.</a> Current opinion in microbiology. 9 (6), 588-594 (2006).</li><li>Ara&#250;jo, D., Henriques, M., Silva, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portrait+of+Candida+Species+Biofilm+Regulatory+Network+Genes.">Portrait of Candida Species Biofilm Regulatory Network Genes.</a> Trends in microbiology. 25 (1), 62-75 (2017).</li><li>Lane, W. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel-plate+flow+chamber+and+continuous+flow+circuit+to+evaluate+endothelial+progenitor+cells+under+laminar+flow+shear+stress.">Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress.</a> Journal of visualized experiments. (59), e3349 (2012).</li><li>Bakker, D. P., van der Plaats, A., Verkerke, G. J., Busscher, H. J., van der Mei, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+velocity+profiles+for+different+flow+chamber+designs+used+in+studies+of+microbial+adhesion+to+surfaces.">Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces.</a> Applied and environmental microbiology. 69 (10), 6280-6287 (2003).</li><li>Zhang, W., Sileika, T. S., Chen, C., Liu, Y., Lee, J., Packman, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+planar+flow+cell+for+studies+of+biofilm+heterogeneity+and+flow-biofilm+interactions.">A novel planar flow cell for studies of biofilm heterogeneity and flow-biofilm interactions.</a> Biotechnology and bioengineering. 108 (11), 2571-2582 (2011).</li><li>Uppuluri, P., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+and+economical+in+vitro+method+for+the+formation+of+Candida+albicans+biofilms+under+continuous+conditions+of+flow.">An easy and economical in vitro method for the formation of Candida albicans biofilms under continuous conditions of flow.</a> Virulence. 1 (6), 483-487 (2010).</li><li>Diaz, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+interaction+between+Candida+albicans+and+commensal+oral+streptococci+in+a+novel+in+vitro+mucosal+model.">Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model.</a> Infection and immunity. 80 (2), 620-632 (2012).</li><li>McCall, A., Edgerton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Approach+to+Flow+Cell+Imaging+of+Candida+albicans+Biofilm+Development.">Real-Time Approach to Flow Cell Imaging of Candida albicans Biofilm Development.</a> Journal of fungi. 3 (1), 13 (2017).</li><li>Zhang, B., Zerubia, J., Olivo-Marin, J. -. 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The authors have nothing to disclose.

Using the flow system as outlined above allows for the generation of quantitative time-lapse videos of fungal biofilm growth and development. To allow for comparisons between experiments it is of critical importance to ensure that the imaging parameters are kept the same. This includes ensuring that the microscope is set up for K&#246;hler illumination for each experiment (many guides are available online for this process). Aside from imaging parameters, there are some important steps to keep in mind when working with the flow apparatus. First, it is important to ensure that the bubble trap is maintained under vacuum during fluid flow, as failure to do so will lead to air being pulled in through the bubble trap. Similarly, when the bubble trap is not under vacuum (i.e., when transporting the flow apparatus) both the inlet and outlet must remain clamped shut; otherwise air will leak in through the PTFE membrane. This clamp does not need to be removed until the bubble trap is once again placed under vacuum. Lastly, it is very important to monitor your flow apparatus for potential clogs or leaks. The most efficient way to check for clogs in your system is to check that there are media dripping from the inlets of the attachment or growth flask, the PD, and the FB. The media from these should be dripping at relatively similar rates if everything is operating smoothly. If a clog is present, you can generally determine the location as the tubing just upstream of the clog will be more rigid.
Once the data have been obtained, we provide numerous ImageJ macros to quantitate the videos. These macros determine multiple parameters of biofilm growth and development, including a measure of the biomass, and the rate that cells attach to and detach from the surface or biofilm. Descriptions of the provided macros are provided below.
Complete analysis performs all the analyses listed below, and automatically outputs the data. This macro can be executed without an open image file while all others require an open video. When executed, it will prompt the user to open an attachment stack file, then a growth stack file. Following this, it will automatically analyze the images and output a data folder containing all the data tables, as described below, to the same folder as the attachment image file. The attachment counter macro is performed only on the attachment file; all other analyses are performed on a concatenated stack of the attachment and growth files. The output data files generated are text files, but should be imported into excel for ease of use.
The sum intensity analysis will analyze each frame of the active window. It adds up all gray values for each pixel that is above the lower threshold designated in step 4.3.7, and outputs one cumulative value per frame. The values generated are proportional to the biomass present within the frame, up until any camera saturation that occurs. The data should then be normalized to the area of the imaging region; this is not performed by the macro.
The coverage area analysis will analyze each frame of the active window for the area of the frame that is covered by cells (above the lower threshold value) as a percentage.
The attachment counter will use frame subtraction to determine the sum intensity of all the cells that attach between each frame. Thus, the first data point is the biomass of the cells that attach between frames 1 and 2; the second data point is the biomass of the attaching cells between frames 2 and 3, etc. These data should be normalized to the area of the imaging region. For easier readability, it is also helpful to integrate this value prior to graphing.
The detachment counter works the same as the attachment counter, but reverses the frame subtraction, such that it determines the sum intensity of the cells that detach between each frame. These data should also be normalized to the area of the imaging region, and integrated prior to graphing. Prior to integration, these data can be further normalized to the total sum intensity of the preceding frame calculated in the sum intensity analysis. This new value represents the proportion of cells that detach from the biofilm at that time point, which is often more valuable data, since the biomass of the detaching cells will increase with increasing biofilm biomass.
While the flow system presented here is more complicated to build and operate than other flow systems, it does offer several advantages. Many of these advantages result from our use of a commercially available channel slide. The tissue culture treatment available for these slides is sufficient to allow Candida cells to adhere to the surface. Additionally, the profile of this channel slide being similar to a traditional slide allows it to easily be used on a wide variety of microscope systems, including upright microscopes using transmitted light at low magnification. Using this type of microscope allowed us to use darkfield microscopy, which made the quantification of data much easier, especially compared to fluorescent microscopy (as there was no photobleaching and low phototoxicity). Traditional microscopy (without optical sectioning), is power conservative, meaning out of focus cells contribute similar numbers of photons to an image as in focus cells of similar size12. This means that, despite our single plane of imaging, the full 3D growing biofilm is still being quantified throughout the experiment, even though the higher regions are out of focus. This single-plane imaging has the advantage of dramatically lowering the phototoxicity damage to cells, but does not provide any information on the 3D architecture of the biofilm. However, this flow system can also be used with fluorescent cells and confocal microscopes to obtain this information13.
The unique two flask recirculating setup of our flow apparatus also has many advantages. First, many flow systems require that the slides be pre-seeded with cells, however our use of a separate cell-seeded attachment flask allows us to image and quantify cells as they adhere to the slide while under flow, and we feel that this is more similar to what occurs in vivo. Additionally, we have previously been able to adjust our microscope for high-speed imaging and image adhesion events as they occurred in real-time, as opposed to quantifying them after the fact11. Second, having a cell-free growth flask that recirculates and can be maintained cell-free over an extended duration allowed us to understand how biofilms grow under flow for over 24 h, a duration that cannot typically be accomplished with non-recirculating systems. We have not yet determined the upper time limit of what can be achieved with our flow system, but we have successfully completed 36 h experiments; however, the longer the experiment, the greater the chance of a leak or clogged filter. Numerous factors can affect the potential duration of an experiment, including the growth rate of the cells, how adhesive they are, and the degree of hyphae formation, making it difficult to define an upper limit on the duration of an experiment. However, if much longer durations are desired than can be achieved with the flow apparatus as presented, the filters can be replaced with an in-line ultraviolet (UV) sterilization box as has been previously described8. This sterilization box may also allow this flow apparatus to be used to image bacteria; our previous attempts to image bacterial strains resulted in rapid clogging of the 0.2 &#181;m filter. Ultimately, we opted not to adopt UV sterilization, as the box is custom fabricated, and as this would result in recirculating dead cells.
Another advantage of this flow system is that it is reasonably inexpensive relative to commercial systems, especially if you need to purchase a microscope with it. In our lab, we were able to purchase a basic transmitted light benchtop microscope and place the entire microscope inside a large standard convection incubator. The only major requirement is that the microscope should have a shutter function (either mechanical or electrical) in order to perform time-lapse microscopy.
While this system is versatile and offers many advantages, it is a low throughput method. Our flow apparatus is unable to grow multiple strains in parallel, unlike other available flow systems. Due to the extensive preparation and cleaning time, we are only able to perform two experiments a week. However, many other flow systems are rather costly, and may clog when Candida cells are grown under hyphae forming conditions.
Additionally, this flow system is quite complex compared to others, and can be difficult to keep in operation. After many experiments, filters begin to clog, tubing begins to wear thin, and parts start to rust or become loose; thus requiring these components to be replaced. The use of filters makes this system incompatible with growth conditions of some fungal strains; in particular, anything that induces flocculation will rapidly clog the 20 &#181;m in-line filter. However, with sufficient experience using the flow system, it becomes easier to detect potential issues before they result in a failed experiment. One thing that can be done to make the everyday operation of the flow apparatus a little simpler is to have a machinist make a replica of the bubble trap housing out of an autoclavable material (such as aluminum or stainless steel), allowing you to autoclave the bubble trap with the rest of the flow apparatus,&#160;as the PTFE membrane and adapter components of the bubble trap are autoclavable.&#160;
In conclusion, the two-phase recirculating flow apparatus presented here represents a unique model to image and quantify in vitro biofilm formation of fungi under flow and in real-time. While the system has its limitations, it is highly adaptable and works well with most microscopes.

Candida albicans (C. albicans) is an opportunistic fungal pathogen of humans that can infect many tissue types, including oral mucosal surfaces, causing oropharyngeal candidiasis and resulting in a lower quality of life for affected individuals1. Biofilm formation is an important characteristic for the pathogenesis of C. albicans, and numerous studies have been done on the formation and function of C. albicans biofilms2,3,4,5, many of which have been conducted using static (no flow) in vitro models. However, C. albicans must adhere and grow in the presence of salivary flow in the oral cavity. Numerous flow systems have been developed to allow for live-cell imaging6,7,8,9,10. These different flow systems have been designed for different purposes, and therefore each system has different strengths and weaknesses. We found that many of the flow systems appropriate for C. albicans were costly, required complex fabricated parts, or could not be imaged during flow and had to be stopped prior to imaging. Therefore, we developed a novel flow apparatus to study C. albicans biofilm formation under flow11. During the design of our flow apparatus, we followed these major considerations. First, we wanted to be able to quantify multiple aspects of the biofilm growth and development in real-time without requiring the use of fluorescent cells (allowing us to study mutant strains and unmodified clinical isolates easily). Second, we wanted all parts to be commercially available with little to no modifications (i.e., no custom fabrication), allowing others to more easily recreate our system, and allowing for easy repairs. Third, we also wanted to allow for extended imaging times at reasonably high flow rates. Lastly, we wanted, following a period of cells attaching to the substrate under flow, to be able to monitor the biofilm growth over an extended time without introducing new cells.
These considerations led us to develop the two-flask recirculating flow system illustrated in Figure 1. The two flasks allow us to split the experiment into two phases, an attachment phase that draws from the cell-seeded attachment flask, and a growth phase that uses cell-free media to continue the biofilm growth without the addition of new cells. This system is designed to work with an incubation chamber for the microscope, with the slide and the tubing preceding it (2 to 5,&#160;Figure 1) being placed inside the incubator, and all other components placed in a large secondary container outside the microscope. Additionally, a hotplate stirrer with an attached temperature probe is used to maintain fungal cells in the attachment flask at 37 &#176;C. As it is recirculating, this system is capable of continuous imaging during flow (can be over 36 h depending on conditions), and can be used on most standard microscopes, including upright or inverted benchtop microscopes. Here, we discuss the assembly, operation, and cleaning of the flow apparatus, as well as provide some basic ImageJ quantitative algorithms to analyze the videos after an experiment.

<table border="0" cellpadding="0" cellspacing="0" style="width:635px;" width="635">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pump</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">07522-20</td>
			<td style="width:229px;">6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pump head</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">77200-60</td>
			<td>6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tubing</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">96410-14</td>
			<td>N/A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bubble trap adapter</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">30704-84</td>
			<td>3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bubble trap vacuum adapter for 1/4&#8221; ID vacuum line</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">31500-55</td>
			<td>3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">In-line filter adapter (4 needed)</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">31209-40</td>
			<td>8,9</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Orange-side Y</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">31209-55</td>
			<td>7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Green-side Y</td>
			<td style="width:71px;">ibidi</td>
			<td style="width:119px;">10827</td>
			<td>2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">* Slides</td>
			<td style="width:71px;">ibidi</td>
			<td style="width:119px;">80196</td>
			<td>4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">* Slide luers</td>
			<td style="width:71px;">ibidi</td>
			<td style="width:119px;">10802</td>
			<td>4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Vacuum assisted Bubble trap</td>
			<td style="width:71px;">Elveflow/Darwin microfluidics</td>
			<td style="width:119px;">KBTLarge - Microfluidic Bubble Trap Kit</td>
			<td>3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Media flasks</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">4980-500</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#181;m air filter</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">431229</td>
			<td>1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Threaded glass bottle for PD and filter flask (2 needed)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">1395-100</td>
			<td>5,10</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ported Screw cap for PD and filter flask (2 needed)</td>
			<td style="width:71px;">Wheaton</td>
			<td style="width:119px;">1129750</td>
			<td>5,10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Screwcap tubing connector</td>
			<td style="width:71px;">Wheaton</td>
			<td style="width:119px;">1129814</td>
			<td>5,10</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tubing connector beveled washer</td>
			<td style="width:71px;">Danco</td>
			<td style="width:119px;">88579</td>
			<td>5,10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing connector flat washer</td>
			<td style="width:71px;">Danco</td>
			<td style="width:119px;">88569</td>
			<td>5,10</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Clamps for in-line filters and downstream Y (7 needed)</td>
			<td style="width:71px;">Oetiker/MSC Industrial Supply Company</td>
			<td style="width:119px;">15100002-100</td>
			<td>7,8,9</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Clamp tool</td>
			<td style="width:71px;">Oetiker/MSC Industrial Supply Company</td>
			<td style="width:119px;">14100386</td>
			<td>N/A</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">20 micron in-line media filter</td>
			<td style="width:71px;">Analytical Scientific Instruments</td>
			<td style="width:119px;">850-1331</td>
			<td>8</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">10 micron in-line media filter</td>
			<td style="width:71px;">Analytical Scientific Instruments</td>
			<td style="width:119px;">850-1333</td>
			<td>9</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 micron inlet media filter</td>
			<td style="width:71px;">Supelco/Sigma-Aldrich</td>
			<td style="width:119px;">58267</td>
			<td>10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">* 0.22 &#181;m media filter</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">SVGV010RS</td>
			<td>11</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">* 0.22 &#181;m media filter &#8220;adapter&#8221;</td>
			<td style="width:71px;">BD Biosciences</td>
			<td style="width:119px;">329654</td>
			<td>11</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rubber stopper</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-131E</td>
			<td>1</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hotplate stirrer with external probe port</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">88880006</td>
			<td>N/A</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Temperature probe</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">88880147</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

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.

Representative images of a normal overnight time-lapse experiment using wild-type C. albicans cells at 37 &#176;C can be seen in Figure 2A and Supplemental Video 1. The images have been contrast enhanced to improve visibility. Quantification of the original data was performed, and representative graphs can be seen in Figure 2B. To generate these graphs, the data were first normalized to the imaging area (i.e., divided by the total imaging area), and the detachment was further normalized to the biomass, as described above. Additionally, the attachment and detachment show the cumulative values over time, rather than the individual frame values generated by the flow biofilm quantification macro. Once the graphs have reached this stage, statistical comparisons can be performed through regression analyses.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58457/58457fig1.jpg" /> 
Figure 1: Schematic of the two-phase recirculating flow apparatus. Connecting black lines indicate tubing, and arrowheads indicate the direction of flow during normal operation. (A) A general schematic overview of the flow system is illustrated. For convenience, the flow system is divided into a green side (upstream of slide) and an orange side (downstream of slide). Bold numbers correspond to parts listed in the Table of Materials. Labels for valves simply mark the location for tubing clamps or hemostats to be placed during experiments. Filter order is as follows: 8&#8211;20 &#181;m inline filter, 9&#8211;10 &#181;m inline filter, 10&#8211;2 &#181;m filter bottle (FB), and 11&#8211;0.22 &#181;m single use disposable filter. Schematic is not to scale. (B) A close-up view of the rubber stopper for the attachment flask, illustrating the four components that pass through the ports: the media outlet, the 0.2 &#181;m air filter that allows gas exchange, the temperature probe (requires drilling an extra hole), and the media return. (C) A close-up view of the pulsation dampener (PD) and the FB, as well as the screw cap assembly used for each port. These bottles need to be air-tight to function. The outlet tubing for the PD should reach deeper into the bottle than the inlet tubing for proper functioning. The gray rectangle in the FB represents the steel filter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58457/58457fig1large.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/58457/58457fig2.jpg" /> 
Figure 2: Candida albicans wild-type cells grown under flow at 37 &#176;C. (A)&#160;Representative darkfield microscopy images of the microcolonies that form under flow at 37 &#176;C at the indicated time points. Scale bar = 100 &#181;m. (B)&#160;Representative image quantification data. The total biomass within the imaging region (determined by densitometry analysis), the cumulative rate of cell attachment, and the percent biomass detachment (detachment rate normalized to the biomass) over time are shown for each strain. Data are means of n &#8805; 3 experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58457/58457fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Video 1" src="/files/ftp_upload/58457/58457video1.jpg" /> 
Supplemental Video 1.&#160;Candida albicans wild-type cells grown under flow at 37 &#176;C. This time-lapse darkfield microscopy video shows the attachment of WT cells to the substrate during the attachment phase (time indicated in the upper left-hand corner; images acquired every 2 min), followed by the subsequent growth and development during the growth phase (starts at 2 h; images acquired every 15 min). Cell-seeded media (1 x 106) were used during the attachment phase, while cell-free media were used during the growth phase. Flow is from the right to left. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58457/JOVE_WT_at_37.wmv" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System at JoVE.com

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

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. ...

real-time-imaging-quantification-fungal-biofilm-development-using-two

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.4K Views.  University at Buffalo. 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.

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

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.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#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.

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

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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 ...

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 ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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

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

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

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

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...