The work was supported by a grant from the National Research Foundation of South Africa (grant number: UID 87903) and the University of the Free State. We are also grateful to services and assistance offered by Pieter van Wyk and Hanlie Grobler during our microscopy studies.

Cryptococcus neoformans and some Acanthamoeba castellanii strains are regarded as biosafety level-2 (BSL-2) pathogens; thus, researchers must take proper precautions when working with these organisms. For example, laboratory personnel should have specific training and personal protective equipment (PPE) such as lab coats, gloves, and eye protection. A biological safety cabinet (level-2) should be used for procedures that can cause infection14.
1. Cultivation and standardization of fungal cells (modified from Madu et al. 15 )
<ol>
	<li>Streak out the test fungal strains (i.e., C. neoformans UOFS Y-1378 and C. neoformans LMPE 046) from stock cultures (no older than 9 months) on yeast-peptone-dextrose (YPD) agar plates. Information on YPD agar&#39;s ingredients can be found in Table 1. 
	NOTE: C. neoformans UOFS Y-1378 has been shown to produce 3-hydroxy fatty acids, while C. neoformans LMPE 046 does not produce 3-hydroxy fatty acids. Refer to Supplemental File 1 for information on how the presence of these molecules is determined.</li>
	<li>Incubate the agar plates for 48 h at 30 &#176;C. 
	NOTE: A plate can be stored for up to 2 months at 4 &#176;C before it can be discarded or used to make a stock culture16.</li>
	<li>Scrape off a loopful of cryptococcal cells (C. neoformans UOFS Y-1378 or C. neoformans LMPE 046) from the 48 h-old plate and inoculate into a 250 mL conical flask containing 100 mL of the chemically-defined YNB broth (6.7 g/L) supplemented with 4% (w/v) glucose. Information on YNB broth&#39;s ingredients can be found in Table 2.</li>
	<li>Incubate the flasks at 30 &#176;C for 24 h while agitating at 160 rpm on a rotary shaker.</li>
	<li>After a 24 h incubation period, count the fungal cells using a hemocytometer and adjust the cell number to 1 x 106 cells/mL with PBS at pH 7.4. 
	NOTE: The prepared C. neoformans UOFS Y-1378 inoculum was used in steps 3.1 and 3.2, while C. neoformans LMPE 046 inoculum was only used in step 3.2.</li>
</ol>
2. Cultivation and standardization of amoeba cells (modified from Madu et al. 15 )
<ol>
	<li>Thaw a stock culture of Acanthamoeba castellanii and bring it to room temperature (RT). 
	NOTE: Amoeba was prepared based on the modified protocols of Axelsson-Olsson et al.8 and Schuster17.</li>
	<li>Pipette 1 mL of the thawed culture and inoculate it into a 50 mL centrifuge tube containing 15 mL of ATCC medium 712. Information on ATCC medium 712&#39;s ingredients can be found in Table 3.</li>
	<li>Manually shake it gently and immediately centrifuge for 5 min at 400 x g and 30 &#176;C.</li>
	<li>Aspirate the supernatant.</li>
	<li>Resuspend the cells in 15 mL of ATCC medium 712 and incubate the tube at 30 &#176;C for 14 days. 
	NOTE: Periodically check the cells, using a simple light microscope, to determine if they are in a trophozoite state. Once they are in a trophozoite state, start a fresh culture.</li>
	<li>Pipette 1 mL from a culture that shows cells in a trophozoite state and use it to inoculate a sterile 50 mL centrifuge tube containing 15 mL of fresh, sterile ATCC medium 712.</li>
	<li>Incubate the tube at 30 &#176;C for 1 week while agitating at 160 rpm on a rotary shaker.</li>
	<li>After a week, count the amoeba cells using a hemocytometer and adjust the cell number to 1 x 107 cells/mL with fresh, sterile ATCC medium 712.</li>
	<li>Perform a viability assay using a trypan blue stain as detailed by Strober18. Proceed further with the cultures that show at least 80% viability.</li>
</ol>
3. Fluorescence staining of cells to study phagocytosis (modified from Madu et al. 15 )
<ol>
	<li>Gathering qualitative data through use of fluorescence microscope 
	NOTE: Perform this assay with Acanthamoeba castellanii and C. neoformans UOFS Y-1378.
	<ol>
		<li>Dispense a 200-&#181;L suspension of standardized amoebae (1 x 107 cells/mL in ATCC medium 712) into chamber wells of an adherent slide and incubate for 2 h at 30 &#176;C for cells to adhere to the surface.</li>
		<li>While amoeba cells are settling down to adhere, stain the standardized C. neoformans UOFS Y-1378 cells that were adjusted to 1 x 106 cells/mL (in 999 &#181;L of PBS) with 1 &#181;L of fluorescein isothiocyanate in a 1.5 mL plastic tube. 
		NOTE: Prepare the stain by dissolving 1 mg of fluorescein isothiocyanate in 1 mL of acetone.</li>
		<li>Gently agitate the C. neoformans UOFS Y-1378 cells on an orbital shaker set at 50 rpm for 2 h at RT and in the dark.</li>
		<li>After 2 h, centrifuge at 960 x g for 5 min at 30 &#176;C to pellet the cells.</li>
		<li>Aspirate the supernatant to remove the PBS with the stain.</li>
		<li>Add 1 mL of PBS to the tube for washing the cell pellet. Wash the cells by gently pipetting.</li>
		<li>Centrifuge the cells at 960 x g for 5 min at 30 &#176;C. Discard the supernatant. Repeat the washing step one more time.</li>
		<li>Resuspend the washed cells in 1 mL of PBS.</li>
		<li>Dispense a 200 &#181;L suspension of the stained C. neoformans UOFS Y-1378 cells to chamber wells containing the unstained amoeba cells.</li>
		<li>Incubate the prepared co-culture at 30 &#176;C for an additional 2 h period. 
		NOTE: The co-culture can be incubated for different time points to suit the purpose of the experiment.</li>
		<li>At the end of the co-incubation period, aspirate the contents of the wells.</li>
		<li>Add 300 &#181;L of PBS to the wells to wash the chamber wells and to remove any unbound co-cultured cells. Do this by gentle pipetting. Aspirate the contents of the wells. Repeat the washing step one more time.</li>
		<li>Prepare the 3% glutaraldehyde solution by adding 3 mL of glutaraldehyde to 97 mL of distilled water.</li>
		<li>Fix the co-cultured cells by adding 250 &#181;L of 3% solution to the chamber wells and incubating for 1 h.</li>
		<li>Aspirate the fixative and wash the chamber wells as detailed from step 3.1.13.</li>
		<li>Dismantle the chamber wells using a tool that was provided with the chamber slides.</li>
		<li>Add a drop of the antifade compound, 1,4-diazabicyclo-[2.2.2]-octane to the slide to prevent auto-bleaching. Cover with a coverslip and seal the sides with a nail polish to prevent evaporation.</li>
		<li>View the co-cultured cells using the 100x objective lens (with oil) of a confocal laser-scanning microscope. 
		NOTE: It is important to take pictures in bright-field and fluorescence to view interaction between amoeba and cryptococcal cells. Wherever possible, the fluorescence can be super-imposed onto the bright-field images. Amoeba cells are typically larger in size (i.e., 45-60 &#181;m), and the trophozoite cells have an irregular shape. Cryptococcal cells are 5-10 &#181;m in diameter and have a globose to ovoid shape. When exposed to a laser, it is possible that unstained amoeba cells may emit auto-fluorescence. Refer to Beisker and Dolbeare19 and Clancy and Cauller20 for methods to reduce autofluorescence.</li>
	</ol>
	</li>
	<li>Acquiring quantitative data through use of fluorescence plate reader 
	NOTE: Perform this assay with Acanthamoeba castellanii and C. neoformans UOFS Y-1378 or C. neoformans LMPE 046.
	<ol>
		<li>Dispense a 100 &#181;L suspension of standardized amoebae (adjusted to 1 x 107 cells/mL in ATCC medium 712) into a black, adherent 96 well microtiter plate.</li>
		<li>Incubate the plate for 2 h at 30 &#176;C to allow amoeba cells to adhere to the surface.</li>
		<li>While amoeba cells are settling down to adhere, stain the standardized C. neoformans UOFS Y-1378 cells that were adjusted to 1 x 106 cells/mL (in 999 &#181;L of PBS) with 1 &#181;L of pHrodo Green Zymosan A BioParticles in a 1.5 mL microcentrifuge tube. Stain C. neoformans LMPE 046 cells as well in a separate tube. 
		NOTE: The dye, unlike FITC, selectively stains cells that are trapped inside the acidic environment of a phagocytic cell21,22. For this technique, it is important to maintain the cryptococcal cells in a medium with a neutral pH (PBS) and amoeba in a medium with a neutral pH (ATCC medium 712). A medium with an acidic environment will result in a false positive reading of the relative fluorescence units, implying that a greater number of cryptococcal have been internalized.</li>
		<li>Gently agitate cryptococcal cells on an orbital shaker set at 50 rpm for 2 h at RT and in the dark.</li>
		<li>After 2 h, centrifuge the microcentrifuge tube at 960 x g for 5 min at 30 &#176;C to pellet the cells. Aspirate the supernatant to remove PBS with the stain.</li>
		<li>Add 1 mL of PBS to the tube to wash the pelleted cells. Wash the cells by gentle pipetting.</li>
		<li>Centrifuge the cells at 960 x g for 5 min at 30 &#176;C. Discard the supernatant. Repeat the washing step one more time.</li>
		<li>Resuspend the pellet of washed cells in 1 mL of PBS.</li>
		<li>Dispense a 100 &#181;L suspension of stained cryptococcal cells to wells containing unstained amoeba cells.</li>
		<li>Incubate the prepared co-culture at 30 &#176;C for an additional 2 h period. 
		NOTE: The co-culture can be incubated for different timepoints to suit the purpose of the experiment.</li>
		<li>At the end of the co-incubation period, measure the fluorescence on a microplate reader. Convert logarithmic signals to relative fluorescence units. 
		NOTE: The dye&#39;s excitation is at 492 nm and emission is at 538 nm. Consult Beisker and Dolbeare19 and Clancy and Cauller20 for methods to reduce autofluorescence.</li>
	</ol>
	</li>
</ol>
4. Use of transmission electron microscopy to study phagocytosis (modified from van Wyk and Wingfield 23 )
<ol>
	<li>Add a 5 mL suspension of amoebae (adjusted to 1 x 107 cells/mL in ATCC medium 712) to a 15 mL centrifuge tube and allow them to settle for 30 min at 30 &#176;C.</li>
	<li>Add a 5 mL suspension of C. neoformans UOFS Y-1378 cells (adjusted to 1 x 106 cells/mL in PBS) to the same centrifuge tube that contains 5 mL of standardized amoeba cells.</li>
	<li>Allow the tube stand for 2 h at 30 &#176;C.</li>
	<li>Centrifuge the tube at 640 x g for 3 min at 30 &#176;C to pellet the co-cultured cells. Aspirate the supernatant. Do not wash the co-cultured cells.</li>
	<li>Fix the co-cultured cells by resuspending the pellet in 3 mL of 1.0 M (pH = 7.0) sodium phosphate-buffered 3% glutaraldehyde for 3 h.</li>
	<li>Centrifuge the tube at 1,120 x g for 5 min at 30 &#176;C to pellet the co-cultured cells. Aspirate the supernatant.</li>
	<li>Add 5 mL of sodium phosphate buffer to the centrifuge tube to wash the pelleted cells. Wash by gently pipetting the contents of the tube for 20 s.</li>
	<li>Centrifuge the tube at 1,120 x g for 5 min at 30 &#176;C to pellet the co-cultured cells.</li>
	<li>Repeat steps 4.8-4.10. Aspirate the supernatant.</li>
	<li>Fix the co-cultured cells again by resuspending the pellet in 3 mL of 1.0 M (pH = 7.0) sodium phosphate-buffered 1% osmium tetroxide for 1.5 h.</li>
	<li>Remove the fixative (osmium tetroxide) by washing the co-cultured cells in a similar manner to removing 3% glutaraldehyde.</li>
	<li>Dehydrate the TEM material (also known as the co-cultured cells) in a graded acetoneseries of 30%, 50%, 70%, 95% and two changes of 100% for 15 min each, respectively. To do so, add 3 mL of the acetone solution to the pelleted cells and let it stand for 15 min. Then, centrifuge at 200 x g for 10 min at RT Discard the supernatant and add the higher percentage of the acetone solution.</li>
	<li>Prepare the epoxy of normal consistency according to the protocol by Spur24. 
	NOTE: The epoxy resin is used for sectioning.</li>
	<li>Embed the TEM material into the freshly prepared epoxy resin. To do so, follow the steps below.
	<ol>
		<li>Add 3 mL of the freshly prepared epoxy to a tube that contains the TEM material resuspended in 3 mL of 100% solution of acetone. Allow the tube to stand for 1 h.</li>
		<li>Centrifuge the tube at 200 x g for 10 min at 30 &#176;C. Aspirate the epoxy-acetone solution.</li>
		<li>Add 6 mL of the freshly prepared epoxy to the pellet in the tube. Allow the tube to stand for 1 h.</li>
		<li>Centrifuge at 200 x g for 10 min at 30 &#176;C. Aspirate all the epoxy-acetone solution.</li>
		<li>Add 3 mL of the freshly prepared epoxy to the tube. Allow the tube to stand for 8 h.</li>
		<li>Centrifuge at 200 x g for 10 min at 30 &#176;C. Aspirate all the epoxy solution</li>
		<li>Add 3 mL of the freshly prepared epoxy to the tube. Keep the TEM material in the epoxy solution overnight in a vacuum desiccator. 
		CAUTION: Epoxy resin is a radioactive material. Use PPE to handle the epoxy resin. The epoxy resin should also be handled in a fume hood. Researchers should follow safety regulations for discarding such material as specified by each country25.</li>
	</ol>
	</li>
	<li>Polymerize the TEM material for 8 h at 70 &#176;C.</li>
	<li>On the ultramicrotome, trim small sections of approximately 0.1 mm x 0.1 mm and 60 nm thickness from the epoxy-embedded material with a mounted glass knife. Assemble sections on a grid and place the grids in a TEM sample holder box before staining.</li>
	<li>Stain the sections with a drop of 6% uranyl acetate for 10 min in the dark. Ensure the sections are completely covered.| 
	NOTE: Reconstitute the stain (6 g) in 100 mL of distilled water. 
	CAUTION: Uranyl acetate is a radioactive material. Use PPE to handle uranyl acetate. Uranyl acetate should also be handled in a fume hood. Researchers should follow safety regulations for discarding such material as specified by each country25.</li>
	<li>Rinse sections by dipping them five times into a beaker that contains 100 mL of distilled water. 
	NOTE: The distilled water should be disposed accordingly as it contains traces of uranyl acetate.</li>
	<li>Stain the section with a drop of lead citrate for 10 min in the dark. Ensure the sections are completely covered. 
	NOTE: Lead citrate should be prepared according to the protocol by Reynold26.</li>
	<li>Rinse sections by dipping them five times into a beaker that contains 100 mL of distilled water.</li>
	<li>Individually assemble the grids with stained sections on a TEM sample holder box.</li>
	<li>View sections with a transmission electron microscope.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+role+of+3-hydroxy+fatty+acids+in+Cryptococcus-amoeba+interactions.">Elucidation of the role of 3-hydroxy fatty acids in Cryptococcus-amoeba interactions.</a> Frontiers in Microbiology. , e765 (2017).</li><li>Hayes, B. K., Heit, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inductive reasoning 2.0. , e1459 (2018).</li><li>Meindl, C., &#214;hlinger, K., Ober, J., Roblegg, E., Fr&#246;hlich, E. <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+fluorescence-based+methods+to+determine+nanoparticle+uptake+by+phagocytes+and+non-phagocytic+cells+in+vitro.">Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro.</a> Toxicology. 378, 25-36 (2017).</li></ol>

The authors declare that they have no competing financial interests.

In the paper, different techniques were successfully employed to reveal the possible outcome that may arise when amoeba interact with cryptococcal cells. Also, we were interested to show the effects of 3-hydroxy fatty acids on the outcome of Cryptococcus-amoeba interactions.
The first technique used was confocal microscopy, which rendered still images. The major drawback of this technique here was that it only gave us information that is limited to a particular timepoint. Any conclusi...

Microbes have evolved over time to occupy and thrive in different ecological niches such as the open physical boundaries of the soil and water, among others1. In these niches, microbes often engage in the direct competition for limited resources; importantly, for nutrients that they use for supporting their growth or space, which they need to accommodate the expanding population2,3. In certain instances, some holozoic organisms like amoeba may even predate on cryptococcal cells as a way of extracting nutrients from their biomass4,5. In turn, this allows such organisms to establish territorial dominance via controlling the population numbers of its prey. Because of this predatory pressure, some prey may be selected to produce microbial factors, such as the cryptococcal capsule6, to reconcile the negative effects of the pressure. However, as an unintended consequence of this pressure, some microbes acquire factors that allow them to cross the species barrier and seek out new niches to colonize7, like the confined spaces of the human body that are rich in nutrients and have ideal conditions. The latter may explain how a terrestrial microbe like Cryptococcus (C.) neoformans can transform to become pathogenic. 
To this end, it is important to study the initial contact that cryptococcal cells may have with amoeba and how this may select them to become pathogenic. More specifically, this may give clues on how cryptococcal cells behave when acted upon by macrophages during infection. It is for this reason that amoeba was chosen as a model for macrophages here, as it is relatively cheap and easy to maintain a culture of amoeba in a laboratory8. Of interest was to also examine how cryptococcal secondary metabolites viz. 3-hydroxy fatty acids9,10 influence the interaction between amoebae and cryptococcal cells.
A simple way of perceiving the interaction between amoeba and its prey with the naked eye is to create a lawn using its prey on the surface of an agar plate and spot amoeba. The visualization of plaques or clear zones on the agar plate depicts areas where amoeba may have fed on its prey. However, at this macro level, only the outcome of the process is noted, and the process of phagocytosis is mechanized cannot be observed. Therefore, to appreciate the process on a cell-to-cell basis, there are several microscopic methods that can be used11,12. For example, an inverted microscope with an incubation chamber can be used to video record a time-lapse of events between a phagocytic cell and its target13. Unfortunately, due to the cost of a microscope with a time-lapse functionality, it is not always possible for laboratories to purchase such a microscope, especially in resource poor-settings. 
To circumvent the above limitation, this study presents a sequential exploratory design that evaluates the interaction of C. neoformans viz C. neoformans UOFS Y-1378 and C. neoformans LMPE 046 with Acanthamoeba castellani. First, a qualitative method is used that precedes a quantitative method. Still images are captured using an inverted fluorescence microscope, as well as a transmission electron microscope to depict amoeba-Cryptococcus interactions. This was followed by quantifying fluorescence using a plate reader to estimate the efficiency of amoeba to internalize cryptococcal cells. When reconciling findings from these methods during the data-interpretation stage, this may equally reveal as much critical information as perusing a phagocytosis time-lapse video.

<table border="0" cellpadding="0" cellspacing="0" style="width:783px;" width="782">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1,4-Diazabicyclo-[2.2.2]-octane</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">D27802</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5-mL plastic tube&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">69715</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-mL Centrifuge tube&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">7252018</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50-mL Centrifuge tube&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">1132017</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">8-Well chamber slide</td>
			<td style="width:176px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">1109650</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Acetone</td>
			<td style="width:176px;">Merck</td>
			<td style="width:123px;">SAAR1022040LC</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">Amoeba strain</td>
			<td style="width:176px;">ATCC&#210;</td>
			<td style="width:123px;">30234TM</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">ATCC medium 712</td>
			<td style="width:176px;">ATCC&#210;</td>
			<td style="width:123px;">712TM</td>
			<td style="width:172px;">Amoeba medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Black 96-well microtiter plate</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">152089</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Centrifuge</td>
			<td style="width:176px;">Hermle</td>
			<td style="width:123px;">-</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Chloroform</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">C2432</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Confocal microscope</td>
			<td style="width:176px;">Nikon</td>
			<td>Nikon TE 2000</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:783px;">Epoxy resin:</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">[1] NSA</td>
			<td style="width:176px;">[1] ALS</td>
			<td style="width:123px;">[1] R1054</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">[2] DER 736</td>
			<td style="width:176px;">[2] ALS</td>
			<td style="width:123px;">[2] R1073</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">[3] ERL Y221 resin</td>
			<td style="width:176px;">[3] ALS</td>
			<td style="width:123px;">[3] R1047R</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">[4] S1 (2-dimethylaminoethanol)</td>
			<td style="width:176px;">[4] ALS&#160;</td>
			<td style="width:123px;">[4] R1067</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Fluorescein isothiocyanate</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">F4274</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Formic Acid</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">489441</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoroskan Ascent FL</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">374-91038C</td>
			<td style="width:172px;">Microplate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glucose</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">G8270</td>
			<td>-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glutaraldehyde</td>
			<td style="width:176px;">ALS</td>
			<td style="width:123px;">R1009</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Hemocytometer</td>
			<td style="width:176px;">Boeco</td>
			<td style="width:123px;">-</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lead citrate</td>
			<td style="width:176px;">ALS</td>
			<td style="width:123px;">R1209</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Chromatography Mass Spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Methanol</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">R 34,860</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Orbital shaker</td>
			<td style="width:176px;">Lasec&#160;</td>
			<td style="width:123px;">-</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Osmium tetroxide</td>
			<td style="width:176px;">ALS</td>
			<td style="width:123px;">R1015</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">pHrodo Green Zymosan A BioParticles</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:123px;">P35365</td>
			<td style="width:172px;">This is the pH-sensitive dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Physiological buffer solution</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">P4417-50TAB</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Rotary shaker</td>
			<td style="width:176px;">Labcon</td>
			<td style="width:123px;">-</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:783px;">Sodium phosphate buffer:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">[1] di-sodium hydrogen orthophosphate dihydrate&#160;</td>
			<td style="width:176px;">[1] Merck</td>
			<td style="width:123px;">[1] 106580</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">[2] sodium di-hydrogen orthophosphate dihydrate</td>
			<td style="width:176px;">[2] Merck&#160;</td>
			<td style="width:123px;">[2] 106345</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Transmission electron microscope</td>
			<td style="width:176px;">Philips</td>
			<td>Philips EM 100&#160;</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Trypan blue&#160;</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">T8154</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ultramicrotome</td>
			<td style="width:176px;">Leica</td>
			<td style="width:123px;">EM UC7</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Uranyl acetate</td>
			<td style="width:176px;">ALS</td>
			<td style="width:123px;">R1260A</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Vacuum dessicator</td>
			<td style="width:176px;">Lasec&#160;</td>
			<td style="width:123px;">-</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Vial</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">29651-U</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">YNB</td>
			<td style="width:176px;">Lasec&#160;</td>
			<td style="width:123px;">239210</td>
			<td style="width:172px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">YPD agar</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">Y-1500</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

complementary use of microscopic techniques and fluorescence reading in studying cryptococcus-amoeba interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Microbes are microscopic organisms that cannot be perceived with the naked eye. However, their impact may result in observable clinically evident illnesses, such as skin infections. When studying certain aspects of microbes, ranging from their morphology, byproducts, and interactions, being able to provide pictorial and video evidence is of the utmost importance.
We first sought to visualize the interaction between cryptococcal ...

Watch this Scientific Journal Video about Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions at JoVE.com

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

complementary-use-microscopic-techniques-fluorescence-reading

Research

JoVE Journal

Immunology and Infection

6.2K Views.  University of the Free State. This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Video: Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...