We thank the Molecular Biosciences (MOBI) doctoral program and the Biology Department at Middle Tennessee State University (MTSU) for providing the funding for this study. The project was also funded in part by a Special Projects grant awarded to D.E.N. by the MTSU Foundation.

NOTE: C. neoformans is a Biosafety Level 2 (BSL-2) pathogen and researchers working with it must take proper precautions. Detailed procedures on how to safely work with BSL-2 pathogens can be found on the Center for Disease Control&#39;s (CDC) website, but it is important to note that all persons coming into contact with C. neoformans should be properly trained in handling pathogenic agents and should always wear appropriate personal protective equipment (PPE), generally latex or nitrile gloves. Further, rotors on centrifuges should be sealed to prevent aerosolization of samples and any spills cleaned up immediately using a 10% bleach solution28.
1. Capsule Induction
NOTE: All steps should be performed at room temperature unless otherwise noted.
<ol>
	<li>Culture C. neoformans in yeast peptone dextrose (YPD) broth (pH 6.5 +/-0.2) and incubate at 37 &#176;C with shaking until they are in log phase (approximately 18 - 36 h).</li>
	<li>Centrifuge cells at 870 x g for 5 min at 21 &#176;C and wash three times with phosphate buffered saline (PBS).</li>
	<li>Use a hemocytometer (or an automated cell counter) to count the cells and resuspend at 2 x 105 cells/2 mL in Dulbecco&#39;s minimal essential media (DMEM).</li>
	<li>To induce the capsule, incubate at 37 &#176;C and 5% CO2 for 18 h. 
	NOTE: The combination of the absence of nutrients in the media and the presence of CO2 stimulates C. neoformans capsule growth.</li>
	<li>Post-incubation, harvest the cells by centrifuging (as above) and removing all but 5-10 &#181;L supernatant. At the same time, harvest a corresponding un-induced cell sample for each strain to be measured. Use these as negative controls. 
	NOTE: The cell pellets will be very small and may be difficult to see. Be careful when aspirating supernatant.</li>
	<li>Stain with either India ink (Section 2.1) or fluorescent dyes (Section 2.2).</li>
</ol>
2. Staining
<ol>
	<li>Staining with only India ink
	<ol>
		<li>To stain the yeast with India ink, resuspend the pellet in the remaining supernatant and add 4&#956;L (approximately half) of cell suspension and 4&#956;L of India ink onto a glass microscope slide. Gently mix and avoid foaming.</li>
		<li>Apply a 13-mm glass coverslip (#1.5 thickness) and seal edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.</li>
	</ol>
	</li>
	<li>Staining with only fluorescent dyes
	<ol>
		<li>To stain the yeast with a fluorescent dye, resuspend the cell pellet in 50 &#181;L PBS with 1% bovine serum albumin (BSA).</li>
		<li>Incubate the culture with an equal volume of Calcofluor white (CFW) (1 &#181;g/mL) and capsule mAb 18B7 (see Table of Materials) conjugated to AlexaFluor488 (AF488) (10 &#181;g/mL) for 30 min at 37 &#176;C.</li>
		<li>Centrifuge cells at 870 x g for 5 min at 21 &#176;C post-incubation and aspirate supernatant.</li>
		<li>Resuspend cell pellet in 10 &#181;L PBS with 1% bovine serum albumin (BSA).</li>
		<li>Pipette 8 &#181;L of cell suspension onto a glass microscope slide.</li>
		<li>Apply a 13-mm glass coverslip (#1.5 thickness) and seal edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.</li>
	</ol>
	</li>
	<li>Staining with both India ink and fluorescent dyes
	<ol>
		<li>To more directly compare the efficacy of India ink staining to that of fluorescent dyes, co-stain the same cell population with both types of stain. To do so, first incubate cells with both a fluorescent capsule and cell wall stain, as per steps 2.2.1 - 2.2.3.</li>
		<li>Next, pipette equal volumes (4 &#181;L) of the fluorescently stained cell suspension and India ink onto a glass slide. Gently mix.</li>
		<li>Apply a 13-mm glass coverslip (#1.5 thickness) and seal the edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.</li>
	</ol>
	</li>
</ol>
3. Image Acquisition/Microscopy
<ol>
	<li>Imaging cells stained with India ink.
	<ol>
		<li>Acquire images using a standard light microscope (100X magnification).</li>
		<li>Image a minimum of 50 cells per condition. 
		NOTE: The number of cells per field will vary, and this is acceptable. It is important, however, that overlapping cells be kept at a minimum, as these are difficult to measure (both manually and with automated software). For these experiments, images were acquired at a depth of 16 bits with exposure times optimized to maximize image contrast while minimizing the blur caused by small movements of the cells.</li>
	</ol>
	</li>
	<li>Imaging cells stained with fluorescent dyes
	<ol>
		<li>To image the cells by fluorescence microscopy, open the microscope imaging software and select the appropriate excitation and emission wavelengths for the fluorophores used (CFW and AF488 are excited using the 405nm and 488 nm lights, respectively). Acquire images using a fluorescence microscope.</li>
		<li>Image a minimum of 50 cells per condition. 
		NOTE: The number of cells per field will vary, and this is acceptable. It is important, however, that overlapping cells be kept at a minimum as these are difficult to measure (both manually and with automated software).</li>
		<li>Acquire a z-stack image series of the cells to ensure that the maximum capsule diameter for each cell within a given field is obtained.
		<ol>
			<li>In the microscope control software, click on the &#34;z-stack&#34; bar to open up the &#34;z-stack&#34; control panel, then select the &#34;First/Last&#34; option in the top left corner of the panel.</li>
			<li>Use the fine-focus control on the microscope to focus at a level that is below the widest point of a single yeast cell and the capsules for all within the current field of view and click &#8220;Set First&#8221;.</li>
			<li>Use the fine-focus control to focus above the widest point of the cell body of the same cell and of the capsule for all of the cells within the current field of view and click &#8220;Set Last&#8221;.</li>
			<li>Then, click &#8220;Start Experiment&#8221; on the &#8220;Acquisition&#8221; tab to acquire the z-stack for the current position. Repeat the process at multiple positions until z-stack images of a minimum of 50 cells have been acquired. 
			NOTE: For these experiments, the confocal pinhole was set to 1.0 AU and an overlapping z-series of 10-20 slices covering 0.7 &#181;M each were acquired at the selected positions. AU = Airy unit.</li>
		</ol>
		</li>
		<li>Next, convert each z-stack series into maximum intensity projections (MIP) using appropriate image analysis software. 
		NOTE: MIPs project the greatest intensity at every plane of the stacked image29. Creating MIPs allows one to produce a 2-D representation of images that correspond to the maximum cell and capsule diameter within the captured 3-D stack.</li>
	</ol>
	</li>
	<li>Imaging cells stained with both India ink and fluorescent dyes
	<ol>
		<li>Acquire images using either a widefield (40X magnification) or confocal microscope (63X or 100X magnification). For cells stained with both India ink and fluorescent dyes, capture images using a widefield microscope equipped with a computer-controlled stage and oil immersion objective. 
		NOTE: The microscope in use should be controlled using an imaging software CFW. fluorescence that is excited through a 340-380 nm excitation filter, and emitted light should be detected through a 435-485 nm barrier filter reflected from a 400-nm dichroic mirror. AF488 fluorescence can be excited through a 465-495 nm excitation filter, and emitted light can be detected through a 515-555 nm barrier filter reflected from a 505-nm dichroic mirror.</li>
		<li>Image a minimum of 50 cells per condition for each staining method.</li>
	</ol>
	</li>
</ol>
4. Manual Measurement of Capsule
<ol>
	<li>For cells stained with India ink or fluorescent stains, use a cell measurement software to manually measure capsule and cell diameter (see Table of Materials). To do this, go to &#34;File&#34; &#62; &#34;Open Image&#34; &#62; &#34;Measure&#34; and use the cursor to draw a straight line through the widest point of the whole cell (total diameter).</li>
	<li>Next, click &#34;Measure&#34; again and draw a straight line through the cell body (cell body diameter). 
	NOTE: Measurements are auto-saved onto the cells.</li>
	<li>Repeat this process for all cells (minimum of 50/group).</li>
	<li>To save images once they are measured, click &#34;File&#34; &#62; &#34;Save As&#34; and name the images appropriately.</li>
	<li>Select the newly measured files and export data to a spreadsheet software.</li>
	<li>Calculate the average capsule diameter by using the equation: ([total diameter] - [cell body diameter]).</li>
</ol>
5. Automated Measurement of Capsule
NOTE: For successful automated measurement, use 16-bit images with a high level of contrast and that are properly focused along with an appropriate image analysis software (see Table of Materials). When imaging C. neoformans for capsule measurement, it is important to focus on the dark ring at the boundary between the cell wall and capsule. This allows the software to correctly delineate the cell and capsule.
<ol>
	<li>Invert and create a copy of all India ink stained cell images using an appropriate image-editing software (see Table of Materials). To do this, click &#34;File&#34; &#62; &#34;Open&#34; to open India ink stained images and then &#34;Control&#34; + &#34;I&#34; to invert the image. Click &#34;File&#34; &#62; &#34;Save As&#34; to save the inverted image.</li>
	<li>To import both the original and inverted images into the software click &#34;File&#34; &#62; &#34;Import Sequence&#34; &#62; &#34;Load Image&#34; &#62; &#34;Define Sequence (Manually)&#34; &#62; &#34;Dimensions (Channel)&#34; &#62; &#34;Import&#34; &#62; &#34;Apply&#34;. 
	NOTE: The C. neoformans cell bodies and capsules can be identified and masked using specific algorithms - referred to as &#39;recipes&#39; - included in the software.</li>
	<li>Use the &#39;Colony Analyzer (Fluorescence)&#39; recipe on the inverted image to detect and mask the cell body, then click &#34;apply&#34;. In the inverted image, the dark ring defining the boundary of the C. neoformans cell becomes brighter than the surrounding capsule.</li>
	<li>Use the &#39;Colony Analyzer (Fluorescence)&#39; recipe on the inverted image to segment the C. neoformans cell bodies from their capsules, then click &#34;apply&#34;. This creates a count mask. Rename this count mask &#34;Cell Body&#34; by right-clicking the tab labeled &#34;count mask&#34;. 
	NOTE: The recipe consists of multiple image processing steps: background removal, object detection, object separation, and subset filtering.</li>
	<li>Next, use the &#39;Cell Proliferation&#39; recipe on the original image to detect and mask the capsule, and click &#34;apply&#34;. This creates a count mask. Rename this count mask &#34;Capsule&#34; by right-clicking the tab labeled &#34;count mask&#34;.</li>
	<li>Using the recipe, &#8216;Capsule Partition&#8217;, create a new mask onto the original image to partition adjacent capsules from one another. To do this, click &#8220;Capsule Partition&#8221; from the recipe dropdown menu. In the Capsule Partition Box, choose the now renamed count mask &#8220;Capsule&#8221; (5.5) for Capsule Region. Choose the now renamed count mask &#8220;Cell Body&#8221; for Crypto Cells (5.4). Choose &#8220;Original&#8221; for the input channel, and &#8220;Create Mask for Partitioned Capsule. Click &#8220;Apply 
	NOTE: The recipe generates cell and capsule diameter, defined as the average of the longest and shortest axes crossing the center of the cell and capsule, and area measurements from detection masks. Find the output that is located under the spreadsheet tab in this software. Repeat this process for all images (minimum of 50 cells/group). The recipe&#39;s parameters could be tuned to optimize detection of individual images or optimized for batch detection of multiple images. Additional measurements can be calculated by the recipe for comprehensive characterization of the cell and capsule&#39;s morphology.</li>
	<li>Export data to a spreadsheet software of choice for further analysis.</li>
</ol>

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The author, Hoyin Lai, is the product manager at DRVision that publishes the automated image analysis software, Aivia.

For decades, the capsule has been a major focus of research for both mycologists and clinicians interested in C. neoformans and cryptococcosis due to its role as a major virulence factor for the pathogen. Using microscopy to measure differences in capsule size between strains and under different growth conditions can provide important information about the pathogen and its responses to various stimuli (i.e., different environmental conditions, potential drug treatments, etc.) Here, we have outlined a method to induce the growth of the capsule in C. neoformans, and compared two different capsule staining protocols. Finally, we showed how image analysis software can be used to automate the measurement of capsule diameter from images of India Ink stained cells, producing results that were comparable to manual measurement methods.
In our experiments, we compared two clinical strains, B18 and B52, with H99S, a standard lab strain of known capsule size18. Both staining methods showed that the clinical strain, B52, had the largest capsule diameter of the three strains tested. However, results from fluorescent measurements were able to discern a difference between the two strains with smaller capsule size where India ink measurements were not. The results from both methods of staining were consistent, suggesting that both are valid but that using images of fluorescently stained C. neoformans may allow for increased sensitivity in measuring strains with the small capsule. Using maximum projections generated from z-stack images of fluorescently stained cells allows researchers to ensure that accurate capsule diameter measurements can be made even when the imaged cells are in different focal planes. These measurements are more difficult for C. neoformans imaged using India ink and standard light microscopy. In any given image, adjacent cells may sit in slightly different focal planes, making it difficult to accurately resolve the cell body-capsule boundary for all cells. This could possibly confound the ability of investigators to detect small changes in capsule size or small differences between strains.
Each method has its own inherent advantages. India ink is the most commonly used by researchers because of its affordability, ease of use, and stability (it doesn&#39;t degrade over time like some fluorescent stains can). Because India ink is a simple, negative stain it can be used in conjunction with standard light microscopes, which are more readily available than fluorescent microscopes. However, if not used in the correct proportions (India Ink:buffer), the stain can create a &#34;feathery&#34; background when imaged, which can complicate automated image analysis. While expensive and more time-consuming to use, fluorescent dyes can be advantageous for their flexibility, and as we show, their sensitivity. They are particularly useful for the measurement of capsule size and cell burden in experiments involving C. neoformans that have been phagocytosed by immune cells and are necessary for the histological examination of C. neoformans capsule in tissue samples (a technique not outlined in this article)32. If staining with fluorescent dyes, such as calcofluor white or fluorescently tagged anti-GXM antibodies, is problematic (e.g., insufficient staining), this can generally be overcome by optimizing the staining protocol (increasing/decreasing the concentration and/or incubation time with the dye).
Regardless of staining methodology, we find that both capsule induction and proper image capture are critical steps in the protocol. The protocol outlined in this paper is one of several standard methods for capsule induction in C. neoformans and has been used successfully in yeast labs for many years23.&#160;C. neoformans capsule induction is CO2-dependent and only occurs under stressful conditions23,33. Therefore, it is critical that it occurs in a 5% CO2 incubator using an appropriate nutrient deficient growth medium. If it is suspected that capsule has not been induced after a standard period of incubation (generally 18-24 h), the cells should be compared to an uninduced control culture that was not exposed to stressful conditions. Comparing the two will determine whether the induction was effective. A successful induction is one in which the majority of cells make a larger capsule, compared to the uninduced control. Because not all of the cells will induce, the cells to be measured should be a heterogeneous representation of the available cells.
The importance of consistent image acquisition cannot be overstated, especially if automated measurement is the goal. Focus, contrast, background fluorescence, as well as the bit-depth and image compression of the files are all important considerations. When possible, large clusters of cells that do not sit in a single focal plane should be avoided, although some budding and touching cells are acceptable and can be unavoidable (this rule should be followed for both manual and automated measuring). This problem can be circumvented to a certain degree when using confocal imaging of fluorescently stained cells. The use of z-stacks to produce maximum intensity projections can enable accurate measurement of cells in different focal planes from a single image29. Additionally, images should be acquired quickly after preparing the sample. This can be particularly important for India Ink stained samples as when they start to dry, the contrast between the background and foreground elements (the cells) can diminish and undesirable background features may be enhanced, which leads to reduced automated segmentation accuracy.
Although capsule diameter measurements are commonplace in the C. neoformans community, up until this point they have typically been performed manually, which requires substantial time and manpower. However, manual measurements are accepted in the field and results can be obtained at little expense. Advancements in image analysis software have made it possible to take the first steps toward automating these measurements. The simple nature of C. neoformans cell body and capsule - appearing as a circle within a circle - in 2D images makes the measurement of their sizes relatively simple for automated image segmentation software packages, without the need to develop novel algorithms. Instead, by linking existing algorithms, C. neoformans cells and capsules can be detected, segmented, and measured. Using this approach, we have automated the measurements of the C. neoformans capsule diameter for cells stained with the most commonly used capsule dye, India ink, and have compared these to manual measurements for the same data sets. This method offers the benefit of reproducibility and removing much of the human error associated with manual measurement while saving the researcher time. Furthermore, additional measurements can be incorporated with minimal effort into the automated analysis workflow. We feel that automated image analysis is a promising approach that allows researchers to measure large numbers of C. neoformans capsule with a high degree of accuracy and efficiency.
Overall, we have demonstrated how to successfully induce capsule growth in C. neoformans, stain the samples with either India ink or fluorescent dyes, and measure capsule diameter using both manual and automated measurements in an image analysis software. Our results suggest that both methods of staining are viable and generally produce consistent results (though there is more variability with India Ink measurements). Further, both manual and automated methods of measurement have the ability to discern varying capsule sizes.

Affecting a quarter million people every year and resulting in more than 180,000 deaths annually, Cryptococcus neoformans is a pathogenic, intracellular yeast and the causative agent of cryptococcosis1,2,3. Hardest hit are HIV-positive patients in poor countries who do not have ready access to antiretroviral therapy, making them acutely susceptible to the illness4,5,6. Data from the CDC indicate that in sub-Saharan Africa, C. neoformans kills more people than tuberculosis annually and more every month than any Ebola outbreak on record1. The most common route of exposure occurs from inhaling desiccated spores that are commonplace in the environment7. Upon entering the lungs, there are several virulence factors that contribute to the success of C. neoformans within infected individuals. The polysaccharide capsule is considered the microbe&#39;s primary virulence factor, as acapsular strains are not virulent8.
The cryptococcal capsule is made of up three principle components: glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs)9. While MPs are a relatively minor cell wall-associated component of the capsule, they are immunogenic and can promote a mostly pro-inflammatory response9,10. In contrast, GXM and GalXM make up the bulk of the capsule (&#62;90% by weight) and have immunosuppressive effects11. In addition to its immunomodulatory effects, the rapid enlargement of the capsule in vivo creates a mechanical barrier to ingestion by host phagocytic cells (i.e., neutrophils and macrophages)12. The C. neoformans capsule and its synthesis are complex, but overall, increased capsule diameter is correlated with increased virulence6,13,14. Given this, it is important for C. neoformans researchers to be able to quickly and accurately quantify capsule measurements.
Both the C. neoformans cell and its polysaccharide capsule are dynamic structures and show changes over time15. The capsule can change in density, size, and assembly in response to changes in the host environment16,17,18. Low iron or nutrient levels, exposure to serum, the human physiological pH, and increased CO2 are known to initiate capsule growth16,18,19,20. Further, researchers have shown structural changes resulting in significant differences in immunoreactivity during an infection, lending an advantage to C. neoformans over its host21,22. This is known because the architecture of the C. neoformans capsule has been analyzed in a variety of ways. Electron microscopy, for example, has revealed that the capsule has a heterogeneous matrix with an inner electron-dense layer underneath an outer, more permeable layer23. Light scattering and the use of optical tweezers have allowed researchers to further elucidate its macromolecular properties24. Analyzing the results from both static and dynamic light scattering measurements, we know that the polysaccharide capsule has a complex branching structure23. Optical tweezers have been used to test the rigidity of the structure as well as evaluate its antibody reactivity24. However, by far the most frequently employed analysis of the C. neoformans capsule is the measurement of its size.
To quantify capsule size, researchers use what should be a simple measurement: the linear diameter of the capsule. Digital microscopes are used to capture images of multiple C. neoformans cells (generally hundreds) stained with either India ink or fluorescent dyes. The size of each cell body and surrounding capsule is measured. The data are compiled, and the average diameter of the capsule is calculated by subtracting the cell body diameter from the whole cell diameter (cell body + capsule). Up until this point, these measurements have been done manually. While generally accurate, this method has drawbacks for researchers. Large data sets can take days or even weeks to analyze by hand. And because these measurements are done manually, subjectivity and human error may affect the result.
Automated computational image analysis has become an indispensable tool for researchers in many areas of molecular cell biology, enabling faster and more reliable analysis of biological images 25,26,27. Precise image analysis techniques are necessary to mine quantitative information from what are often complex and immense data sets. However, some measurements, especially the measurement of C. neoformans capsule, have been difficult to automate. Accurately identifying the interface between the cell wall and capsule, which generally appears as a dark ring when imaged by phase-contrast microscopy, can be troublesome to resolve using a simple threshold. Further, C. neoformans cells in culture tend to clump together and accurate segmentation of the cells is necessary for accurate measurements.
The aim of this project was to (i) illustrate one of the standard protocols for capsule induction in C. neoformans, (ii) compare and contrast India ink and fluorescence staining as they pertain to capsule diameter measurements, (iii) develop simple, computational methods to measure capsule diameter using images of India ink stained cells using an image analysis software, and, (iv) assess the benefits and limitations of measuring capsule diameter manually and using software automation. We find that of the two staining methods, fluorescent labeling of the cell wall and capsule, while more time-consuming, provided the most consistent results between experiments. However, both methods enabled us to successfully distinguish between lab and clinical C. neoformans strains exhibiting different capsule sizes. Further, we were able to automate the measurement of capsule diameter from India ink stained images and found that this was a viable alternative to manual measurement of capsule.

<table border="0" cellpadding="0" cellspacing="0" width="983">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Capsule Induction</td>
			<td style="width:204px;"></td>
			<td style="width:117px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C. neoformans cells</td>
			<td></td>
			<td></td>
			<td>The clinical lab strain, H99S, was a kind gift from Dr. John Perfect (Duke University).&#160; The clinical strains, B18 and B52, were kind gifts from Dr. Greg Bisson (University of Pennsylania).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Peptone Dextrose Broth (YPD)</td>
			<td>Fisher Scientific</td>
			<td>DF0428-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>This is made in the lab using standard recipe (137mM NaCl, 2.7 mM KCl, 10mM Na2HPO4O, 2 mM Kh2PO4O)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/high-glucose with L-glutamine, without sodium pyruvate</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Falcon</td>
			<td>CL5335-5EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Thermo Scientific&#160;</td>
			<td></td>
			<td>MaxQ6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Isotemp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Legend XTR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Legend Micro 21R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">India ink</td>
			<td>Fisher Scientific</td>
			<td>14-910-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcofluor white</td>
			<td>Sigma-Aldrich</td>
			<td>18909-100ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18B7 mouse anti-GXM antibody conjugated to Alexafluor 488</td>
			<td></td>
			<td></td>
			<td>A kind gift from Dr. Arturo Casadevall (Johns Hopkins University)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with 1% Bovine Serum Albumin (BSA)</td>
			<td></td>
			<td></td>
			<td>PBS is the same recipe listed above (line 4) with 1% BSA added and filter sterilized.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>Corning</td>
			<td>2855-18</td>
			<td>#1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clear nail polish or other non-toxic sealant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image Acquisition&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil</td>
			<td>Cargille&#160;</td>
			<td>16484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope with immersion oil objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio A1 with a Plan - NEOFLUAR 100x oil immersion NA 1.30 objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope camera</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axiocam ErCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope with oil immersion objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 700 laser scanning confocal equipped with a Plan-Apochromat 63X NA 1.4 oil immersion DIC M27 objective.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope software</td>
			<td>Zen 2009</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope camera</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon Ti-Eclipse with a Intensilight epifluorescence illuminator (Nikon), CoolSNAP MYO microscope camera (Photometrics), Plan Apo 60x NA 1.40 oil immersion objective (Nikon) and 1.5x magnification changer.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Widefield imaging software</td>
			<td>Nikon Elements (Nikon)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capsule Measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image editing software</td>
			<td>Photoshop (Adobe)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope software for manual measurement</td>
			<td>Axiovision (Carl Zeiss)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image analysis software for automated meesurement</td>
			<td>Aivia (DRVision Technologies)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spreadsheet software</td>
			<td>Excel (Microsoft)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

size matters: measurement of capsule diameter in cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

To illustrate capsule induction, cell staining, imaging, and measuring techniques, we used three strains of C. neoformans: the common, well-characterized laboratory strain, H99S30, and two clinically isolated strains of previously unknown capsule diameter, B18 and B5231.
The workflow of capsule induction, staining, and image acquisition using India ink is shown in Figure 1A. Images taken from all three strains are shown in Figure 1B both pre- and post-induction. The researcher will know if the capsule induction was a success by comparing the cells that have undergone capsule induction to those from the same culture that have not. A clear size increase is generally apparent in most strains post-induction, although some strains will exhibit naturally small capsules even after induction (see images of B18 in Figure 1B). It should be noted that capsule induction will fail if residual rich media is not removed prior to induction or if CO2 is not present. In these cases, only modest (if any) increases in capsule size will be observed. Diameters from all three strains were measured manually and the results are shown in Figure 1C. In two of the three experiments, there was no difference in size between H99S and B18. However, a significant difference in size was consistently observed between the largest strain, B52, and H99S and B18 (p &#60;0.001 and p &#60;0.001, respectively, standard least squares test with simple contrasts, sample size = 50 cells/strain).
Figure 2A depicts the workflow of capsule induction, staining, and image acquisition using two fluorescent dyes, one for the capsule (18B7-AF488) and one for the cell wall (Calcofluor White). Images were captured as z-stacks for all three strains post-induction, ensuring that the maximum capsule and cell body diameter was captured for each cell to be measured (Figure 2B). Each z-stack was processed to produce maximum intensity projections, compressing the 3-dimensional stack into a 2D image prior to analysis (Figure 2C). These were then measured manually (Figure 2D). Unlike the measurements for India ink stained cells, the fluorescent staining enabled us to discriminate between all three strains on the basis of their capsule diameter in all three repeats of the experiment. Here, we found that B18 had the smallest capsule diameter and B52 the largest (p &#60;0.001 for all comparisons, standard least squares test with simple effects, sample size = 50 cells/strain).
To further assess the two methods of C. neoformans capsule staining, measurements from all three strains were compared. There was no significant difference between the capsule sizes determined using the two methods of staining (Figure 3A) (multivariate ANOVA with simple effects, sample size = 50 cells/strain). This conclusion was consistent with the findings of a separate experiment where the lab strain, H99S, was co-stained with India ink and fluorescent dyes (Figure 3B-C). However, there was greater variability between experiments using India ink, as evidenced by the fact that only one out of three experiments showed a significant difference in capsule size between H99S and B18, compared to the significant difference observed between these strains in all three fluorescent experiments (Figure3D-E) (multivariate ANOVA with simple effects, sample size = 50 cells/strain).
India ink stained images that meet the criteria listed in the protocol (section 5) can be measured using an automated system. The workflow shown in Figure 4A guides the user through the steps necessary to utilize the recipes designed to measure the C. neoformans capsule. After developing the workflow, it was validated by measuring a cache of existing images of C. neoformans cells by three researchers using both manual and automated methods. There were no significant differences between the researcher&#39;s measurements when made manually, and this was also true when the measurements were made using automated image analysis software (Figure 4B) (ANOVA, sample size = 50 cells/researcher). However, there was a small but significant difference in the capsule size reported between the two methods (Figure 4C) ANOVA, sample size = 150 cells/group. Capsule diameter was consistently slightly smaller when measured via automation than manual measurements (0.3 &#181;m, p &#60;0.001). Additionally, when comparing between capsule size measurements made by separate researchers from the same image sets, the standard error was smaller when image analysis software was used for quantification instead of manual methods, which is suggestive of more precise, reproducible measurements using the automated method. For successful automated measuring, cells must be in focus, have a medium to large capsule, and not be overly clustered (Figure 4D). We found that poorly focused images of clustered cells with small capsules could not be automatically measured in the software. However, we assert that these may also be challenging to measure accurately using manual methods.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57171/57171fig1.jpg" /> 
Figure 1: India ink staining to measure C. neoformans capsule. (A)Schematic representation of C. neoformans capsule measurement by India ink staining. In brief, capsule expression is induced using nutrient deprivation and exposure to CO2, the cells are resuspended in India ink and mounted on glass slides. Images of stained cells are acquired by light microscopy, with fields either selected by the user or by tile-scanning using a computer controlled stage. Images can either be analyzed manually or processed for automated image segmentation and analysis. (B) Images of the common lab strain, H99S and two clinical strains, B18 and B52, both pre- and post-capsule induction. Scale bars represent 10 &#181;m (C) Graph of a representative experiment showing capsule diameter of all three strains stained with India ink and measured manually (error is represented as standard error (SE)). Statistical significance is indicated as follows: *, p &#60;0.05; **, p &#60;0.01; and ***, p &#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig1large.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/57171/57171fig2.jpg" /> 
Figure 2: Fluorescent staining to measure C. neoformans capsule. (A) Schematic representation of C. neoformans capsule measurement by Calcofluor White (CFW) and 18B7-AF488 co-staining. After capsule induction and staining, the cells are mounted and imaged by laser scanning confocal microscopy. As cells may be in different focal planes, z-stacks are acquired at each position. These are processed to produce maximum intensity projections that can be used to accurately measure the maximum capsule diameter for each cell. (B) Images of the common lab strain, H99S and two clinical strains, B18 and B52, post-capsule induction. Note: Scale bars represent 10 &#181;m for all images. (C) Maximum intensity projections of fluorescently labeled C. neoformans cells. (D) Graph of a representative experiment showing manually measured capsule diameter of all three strains stained with CFW and 18B7-AF488 (error is represented as standard error (SE)). Statistical significance is indicated as follows: *, p &#60;0.05; **, p &#60;0.01; and ***, p &#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig2large.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/57171/57171fig3.jpg" /> 
Figure 3: Comparison of results between India ink and fluorescent staining. (A) Analysis of three C. neoformans strains stained with India ink or fluorescent dyes, n = 3 (error is represented as standard error (SE)). (B) Images of a single C. neoformans cell stained with India ink and both fluorescent dyes (scale bar represents 13 &#181;m). Horizontal lines demarcate the edges of the capsule and vertical lines demarcate the edges of the cell body. (C) Quantification of a representative experiment of H99S co-stained with both India ink and fluorescent dyes (error is represented as standard error (SE)). (D,E) Depiction of capsule diameter variability between India ink experiments (D) and fluorescent experiments (E). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig3large.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/57171/57171fig4.jpg" /> 
Figure 4: Computational analysis of C. neoformans capsule diameter from images of India ink stained cells. (A) Schematic representation of image segmentation and C. neoformans capsule measurement using an automated measurement technique (see Table of Materials). (B) Comparison of capsule measurements performed by 3 separate investigators using manual (Manual 1-3) and automated methods (Automated 1-3). Each investigator used an identical cache of C. neoformans images by both methods (error is represented as StDev). (C) Combined average of manual measurements and automated measurements (error is represented as standard error (SE)). Examples of both (D) ideal and (E) non-ideal images of C. neoformans. Non-ideal images cannot be successfully measured in the software. Statistical significance is indicated as follows: *, p &#60;0.05; **, p &#60;0.01; and ***, p &#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans at JoVE.com

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

size-matters-measurement-capsule-diameter-cryptococcus

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.4K Views.  Middle Tennessee State University. The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Video: Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...