The authors acknowledge Dr. Maryam Hussain for previous work on this method, the group of PD Dr. Kerstin Danker (Charit&#233;-Universit&#228;tsmedizin, Berlin) for providing the THBMECs and Juliane Weber for critically reading the manuscript. This study was supported by the RTK 2155 (ProMoAge).

1. Preparation of Buffer and Reagents
<ol>
	<li>Prepare 10x phosphate buffer saline (10x PBS) by adding 80 g of sodium chloride (NaCl), 2 g of potassium chloride (KCl), 14.4 g of disodium-hydrogen-phosphate dihydrate (Na2HPO4 &#183; 2H2O) and 2 g of potassium-dihydrogen phosphate (KH2PO4) in a 1 L glass flask in 1 L of double distilled H2O. Autoclave the 10x PBS solution and dilute 100 mL of this solution in 900 mL of double distilled water to get 1x PBS.
	<ol>
		<li>Use the autoclave to sterilize solutions. Put the glass flask into the basket, close the lid and sterilize it for 15 min at 121 &#176;C and 98.9 kPa. 
		NOTE: This protocol is always used for autoclaving solutions in further steps.</li>
	</ol>
	</li>
	<li>Prepare a 10 &#181;g/mL collagen IV and 10 &#181;g/mL fibronectin solution by diluting 0.5 mg/mL fibronectin solution and the 0.3 mg/mL collagen IV solution each with 1x PBS to 100 mg/&#181;L aliquots in 1.5 mL micro tubes. Thereafter, mix 100 &#181;L of both aliquots with 1,800 &#181;L of 1x PBS in 2 mL micro tubes and store them at -20 &#176;C.</li>
	<li>Prepare DMEM/F-12 medium by adding 4% fetal bovine serum and 2 mM L-glutamine and 100 mg/L penicillin/streptomycin to the medium and store it at 4 &#176;C.</li>
	<li>Prepare 1x trypsin-EDTA solution by diluting 5 mL of the 10x concentrated trypsin-EDTA solution with 45 mL of 1x PBS in a 50 mL tube and store it at 4 &#176;C.</li>
	<li>Prepare 500 mL of LB medium by weighing 10 g of LB broth base in a 500 mL glass flask. Add 500 mL of sterilized water and autoclave it.</li>
	<li>Prepare LB agar by weighing 10 g of LB broth base and 7.5 g of agar-agar in a 500 mL glass flask. Add 500 mL of sterilized water before autoclaving and do not close the lid of the flask. Autoclave and leave the solution to cool down until it is warm to the touch.</li>
	<li>Prepare antibiotic-free medium by adding 4% fetal bovine serum and 2 mM L-glutamine, but no penicillin/streptomycin to the DMEM/F-12 medium and store it at 4 &#176;C as in step 1.3.</li>
</ol>
2. Growth of the Blood-brain Barrier Mimicking Cells
<ol>
	<li>To assemble the 12 well plate, put the cell culture inserts into the wells (Figure 2A).
	<ol>
		<li>Unpack the plate and each insert in a biological safety cabinet and perform further steps there. Use sterilized forceps to grab the insert at its broad base to move it.</li>
	</ol>
	</li>
	<li>Coat the porous membrane of each insert with 90 &#181;L of 10 &#181;g/mL collagen IV and 10 &#181;g/mL fibronectin mixture. Incubate the 12 well plate for 24 h at 37 &#176;C in a cell culture incubator (Figure 2B).</li>
	<li>Wash the inserts twice by pipetting 1 mL of 1x PBS into each insert and aspirate the solution with a vacuum pump for cell cultures.</li>
	<li>Equilibrate the membranes by pipetting 0.5 mL of prewarmed DMEM/F-12 medium in the upper and 1.5 mL in the lower chamber. Incubate the plate for 30 min at 37 &#176;C in a cell culture incubator with 5% CO2 atmosphere (Figure 2C).</li>
	<li>Seed 2 x 105 human microvascular endothelial cells into each upper chamber and incubate the 12 well plate at 37 &#176;C in a cell culture incubator (Figure 2D).
	<ol>
		<li>Aspirate the medium from the cell culture flask using a vacuum pump for cell cultures and wash the monolayer by pipetting 10 mL of 1x PBS and aspirating the solution afterwards with a vacuum pump.</li>
		<li>Cover the cells completely with 1x concentrated trypsin-EDTA by pipetting 5 mL of the solution into the cell culture flask. Incubate the flask for 3-5 min at 37 &#176;C in a cell culture incubator. 
		NOTE: If the cells are not dissociated, firmly tap the flask against the palm of the hand to dislodge the cells.</li>
		<li>Take 5 mL with a pipette as an aliquot from the cell suspension in a 15 mL tube and add 5 mL of the FCS-containing medium to stop the enzymatic reaction.</li>
		<li>Centrifuge the suspension for 3 min at 210 x g, remove the supernatant with a vacuum pump, and resuspend the pellet in 5 mL of medium with a pipette.</li>
		<li>Use the cell counter by mixing 10 &#181;L of the cell suspension with 10 &#181;L of 0.4% trypan blue stain in a 1.5 mL micro tube. Add 10 &#181;L of the mixture into a counting chamber slide, put it into the cell counter, and start counting.
		<ol>
			<li>Focus the counter on the cells so that their edge is dark blue and the middle white. Thereafter, start the appropriate program for cell counting.</li>
		</ol>
		</li>
		<li>To calculate the volume for each insert, divide the obtained 2 x 105 cells per insert with the calculated concentration of the cell suspension.</li>
	</ol>
	</li>
</ol>
3. Cultivation of the Blood-brain Barrier Model
<ol>
	<li>Incubate the 12 well plate for 14 days at 37 &#176;C.</li>
	<li>Change 0.5 mL of medium for the upper chamber and 1.5 mL of medium for the lower one every 2-3 days. Warm the medium before putting it in the cell flask. Aspirate with the vacuum pump (Figure 2E). 
	NOTE: Work carefully to avoid touching the membrane.</li>
	<li>Check the status of the cells by imaging them with a microscope and determine the confluency. Ensure that the confluency is 100% after 14 days.</li>
</ol>
4. Preparation of Bacteria
<ol>
	<li>A day before measurement, put a colony of the E. coli strain GM2163 into LB medium. Incubate the culture tube for 24 h at 37 &#176;C with 180 rpm in an incubation shaker (Figure 2F).
	<ol>
		<li>Cultivate the E. coli strain on an LB agar plate at 4 &#176;C. Take one colony with a sterilized pick and put the pick in the prepared culture tube with 3 mL of LB medium.</li>
		<li>Prepare an LB agar plate for each insert with warm LB agar solution and fill the Petri dishes to half their total volume. Let them become solid and store them at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Treatment of Cells
<ol>
	<li>On day 14 after seeding, treat cells with compounds or measure the transendothelial electrical resistance (TEER), if planned (Figure 2G). 
	NOTE: Always have some untreated cells as a control.
	<ol>
		<li>To treat cells with the compound of interest, dilute the compound to the final concentration in DMEM/F-12 medium. Add 0.5 mL of this mixture into the upper chamber and 1.5 mL into the lower chamber. Incubate the plate in a cell culture incubator for the desired time.</li>
	</ol>
	</li>
	<li>Afterwards, exchange the complete medium with antibiotic-free medium by aspirating with the vacuum pump and pipetting (Figure 2H).</li>
</ol>
6. Measurement of Permeability
<ol>
	<li>To get constant concentrations of bacteria, measure the optical density with a photometer at a wavelength of 600 nm. Dilute the overnight solution of bacteria with LB medium in a 50 mL falcon tube to an OD600 of 0.5 &#177; 0.05. Work on ice.
	<ol>
		<li>Fill 1 mL of LB medium into a cuvette. Start the photometer and put the cuvette in, marked side forward. Press the bottom &#34;Blank&#34; for measuring the blank value afterwards.</li>
		<li>Measure the density of the bacterial solution by filling it into a cuvette, putting it in, and pressing the bottom sample. Repeat the measurement during the diluting until obtaining the final concentration.</li>
	</ol>
	</li>
	<li>Work in a biological safety cabinet where bacteria can be handled with the prepared 12 well plate and bacterial solution at OD600 = 0.5. Add 450 &#181;L of bacterial solution only into each upper chamber containing 0.5 mL of medium (Figure 2I).</li>
	<li>Incubate the 12 well plate for 6 h at 37 &#176;C in an incubator. 
	NOTE: The protocol dictates to pause here.</li>
	<li>Sample 50 &#181;L of the medium with a pipette from each lower chamber by removing the insert with forceps (Figure 2J). Take care not to spill the medium from the upper chambers to the lower ones.</li>
	<li>Plate each sample on a separate agar-plate (Figure 2K). Drop the sample on the plate and streak out the solution with a cell spreader.</li>
	<li>Incubate the agar plates for 24 h at 37 &#176;C in an incubator.</li>
	<li>Count the colonies in each plate (Figure 2L).</li>
</ol>
7. Analyzing Data
<ol>
	<li>Write the data in a table and calculate the average and standard deviation of the observed colonies of treated and untreated cells.</li>
	<li>Display the average as the absolute number of colonies.
	<ol>
		<li>To normalize the results, calculate the relative number of colonies by dividing all results with the control value.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60692/60692fig2a.jpg" /> 
Figure 2: Detailed presentation of the individual steps in the protocol. (A) Put the inserts with sterilized forceps into the 12 well plate. (B) Coat each insert with 90 &#181;L of fibronectin and collagen IV mixture and incubate for 24 h. (C) Equilibrate the membranes with prewarmed medium for 30 min. (D) Seed 2 x 105 human brain microvascular endothelial cells per insert. (E) Cultivate the plate for the appropriate amount of time. (F) One day before measuring, put an E. coli colony into a LB medium culture tube and incubate for 24 h. (G) Treat cells or measure TEER. (H) Exchange complete medium with antibiotic-free medium. (I) Add 450 &#181;L of bacterial solution (OD600 = 0.5) into each upper chamber and incubate for 6 h. (J) Sample 50 &#181;L of medium from each lower chamber removing insert with forceps. (K) Plate the sample on agar plates and incubate for 24 h. (L) Count the colonies and analyze the data. <a href="https://www.jove.com/files/ftp_upload/60692/60692fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

&#160; &#160;

The authors have nothing to disclose.

Limited insight into the pathogenesis of microbial traversal limits further development of therapies for POD or meningitis. The mortality and morbidity of these diseases demand better patient treatment, require research of the underlying mechanisms, and need a robust platform for compound screening. The multifactorial events can be studied with human BMECs. Several successful reported isolation procedures of BMECs from a number of species have shown a loss of the cells&#39; characteristics molecular signature52,53. The described THBMECs in this procedure were transfected in very early passages, where they exhibited specific brain endothelial cell characteristics and preserved them43. This is important, because not all steps in the affected pathways have been discovered so far, and this model seems to mimic conventional BMECs. Our presented model shows direct influences on BMECs and the microbial traversal through the BBB.
The handling of THBMEC cells is straightforward, and the required technical equipment exists in most life science laboratories. Our model allows for an immediate start of investigative procedures after the THBMECs have built a tight monolayer. The field of applications can be extensive because of the possible combinations between new tests and conventional assays such as TEER measurement or labeling with tracers54. It is also possible to add astrocytes or pericytes to make a co- or triple-culture model. The influence of drugs on the microbial traversal could also be tested in our model by treating the THBMECs with compounds before inoculating the upper chamber with bacteria. In fact, it is possible to purchase inserts with filters for 96 well plates allowing the automation of the procedure. This can facilitate the implementation of high-throughput drug screening systems to accelerate the discovery of drugs against the mentioned diseases and to reduce side effects on the BBB during drug development.
A critical step in the presented method is the incubation time after adding the bacteria to the upper chamber. It is important to use hours as timelines in the protocol, because the generation time of E. coli is only 20 min55. Otherwise, use of different time points could lead to misleading results. There is also a possible risk of contamination between upper and lower chamber during the bacterial exposure if the plates are not handled with care. Any alterations to the 12 well plate at this point could contaminate the medium in the lower chamber.
E. coli is one well-known, very common cause of bacterial meningitis. Further investigations should test different bacteria that are also associated with meningitis, such as Neisseria meningitidis56 or Streptococcus pneumoniae57. These seem to use different mechanisms to cross the BBB and need to be better understood for the treatment of patients. In elderly patients, the incidence for POD increases26 as well as the number of occurring comorbidities. It is known that there are interactions between different diseases, especially systemic ones like diabetes. In our model, it is possible to simulate those conditions or treat the cells before adding the bacteria.
The model is limited by the direct contact of THBMECs and bacteria, and further research is necessary to investigate potential mechanisms of contact to detect the involved pathways and proteins. However, it is possible to remove inserts and harvest the cells for further analysis. The TEER of the model is lower in comparison to stem cell models38,39,40. We confirmed this by using a bacterial concentration that did not cross the BBB in untreated cells after 6 h.
In summary, this method represents a robust platform to analyze the traversal of bacteria through the BBB with the potential to expand it for high-throughput drug screenings.

The human BBB is a unique boundary of brain tissue, separating the brain from the blood. It strictly regulates the passage of larger and hydrophilic molecules, blocks paracellular diffusion, and maintains brain homeostasis. It also protects the brain from plasma fluctuations, toxins, microbes, and guides inflammatory cells as part of the central nervous system (CNS) immunity. Since its discovery a century ago1, many studies have been carried out to understand the structure and function of the BBB. The complex interactions of cells, proteins, and signals from brain and blood demand still further investigation and models.
The human BBB is composed of three cell types: brain microvascular endothelial cells (BMECs), pericytes, and astrocytes2,3. The BMECs differ from the majority of the endothelial cells in the body in that they possess a high number of tight junctions and adherens junctions4, low pinocytotic activity2,5, and a continuous basement membrane6,7 to block paracellular diffusion. Small lipophilic molecules can diffuse and pass the BBB following their concentration gradient; larger and hydrophilic molecules enter or leave the brain only through polarized expressed selective transport systems8. This regulation results in a high transendothelial electrical resistance (TEER) of 1,500-2,000 &#937;&#183;cm2 that is inversely correlated to permeability9,10. Although BMECs build a tight barrier, they can react to local and peripheral signals11,12. There is a close interaction between BMECs and astrocytes13; the astrocyte end-feet build a layer around the vessels and induce the formation of tight junctions13,14. They are involved in BBB maturation with different factors, including transforming growth factor-&#946; (TGF-&#946;)15,16. In addition, pericytes play a key role in the regulation of angiogenesis17 and preventing apoptosis of the endothelium in cellular differentiation18 (Figure 1). They are embedded in the basement membrane and provide structural stability of the vessel wall19.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60692/60692fig1.jpg" /> 
Figure 1: Schematic structure of the blood-brain barrier. The unique structure of the human BBB is composed of three different cell types. The microvessel lumen is surrounded by endothelial cells, which are enriched in tight junctions, and are not fenestrated. They are embedded in the basement membrane, like the pericytes. These cells are important for structural stability of the vessel wall and play a role in the development of the BBB next to the astrocytes. Their end-feet build a close layer around the vessel and support the building of tight junctions. All components of the BBB are important for physiological functionality. <a href="https://www.jove.com/files/ftp_upload/60692/60692fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Many different pathologies are related to the collapse of the BBB (e.g., septic encephalopathy). The affected patients have increased protein levels in the cerebrospinal fluid20, and the brain parenchyma in affected rodents shows an increased uptake of marked colloidal iron oxide and amino acids21,22. These results point towards an increased permeability of the BBB that occurs alongside an increased pinocytosis in BMECs21 and endothelial activation23. Another associated pathology related to an altered BBB is meningitis, a medical emergency and a complex inflammation accompanied with cerebral edema that can lead to neuronal cell death. The primary entry site of circulating bacteria is supposed to be the microvessels24; however, the BBB prevents the entry of bacteria. The permeability of the BBB is not always linked to an increase in experimental hematogenous meningitis25 and the mechanisms can be multifactorial. Coincidence of sepsis with postoperative delirium (POD)26 and the association with preoperative infections27,28 indicates the need for a BBB model that enables the direct exposure to bacteria to get a better understanding into bacterial pathogenesis.
There are many gaps in understanding and quantifying the microbial traversal through the BBB. Therefore, we developed a model that allows a convenient testing of different factors and conditions with a direct correlation between bacterial traversal and influences on the permeability of the BBB. Previous work focused on the paracellular permeability and included TEER measurement and tracer flux. In addition, macromolecule transport was analyzed by conjugated molecules or antibodies, whereby different models using only endothelial cells or combinations with astrocytes and pericytes were developed. Due to the difficulty in obtaining human tissue on a regular basis, many animal-based models are used. Brain endothelial cells of bovine and porcine origin form tight monolayers with a high TEER that form well-shaped apical-basal polarity and are suited for investigations of small molecule transport through the BBB. The proteins differ in sequence from their human homologues29,30, making investigation of therapeutic antibodies difficult. For this reason, murine or human culture models may be preferable. Mouse or rats as sample sources have the advantage of being obtained from well characterized species but yield few cells for study purposes. This can be circumvented by the use of immortalized mouse brain endothelioma (END) cell lines bEND.3, bEND.5 or cEND31,32,33.
Primary cultured cells from human tissue are difficult to obtain and to handle on a regular basis. Therefore, most human cellular models used in research investigating the human BBB are immortalized endothelial cell lines. A published cell line is human cerebral microvascular endothelial cell line hCMEC/D3, which is well suited for studying drug uptake and is easy to handle. The cells build a monolayer and express the characteristic tight junction proteins of the BBB34, whereas the expression level of claudin-5 is reported to be lower than in intact microvessels35 and many specific transporters have been detected at transcript level36 as well as in proteomic studies34. A relatively low TEER in the range of 30-50 &#937;&#183;cm2 is still a challenge37. Another source for brain endothelial cells are human pluripotent stem cells (hPSCs)38 and human cord blood-derived stem cells of circulating endothelial progenitor and hematopoietic lineages39,40. Both protocols of differentiation result in tight cell monolayers and high TEER values (e.g., 1,450 &#937;&#183;cm2 in co-cultures)38. These stem cell models require extreme care for cultivation, yet offer the opportunity to study the influence of regulating hormones41 or diseases with genetic backgrounds42 on BBB development.
In this study, we established an immortalized transfected human brain microvascular endothelial cell line, THBMEC43, to mimic the BBB and to study bacterial traversal. Cells are seeded on a filter and grown to 100% confluency in this cell culture model. Bacteria are inoculated in the upper part of the cell culture chamber. We use Escherichia coli (E. coli) in our sample study because of the high incidence of E. coli meningitis44. It has been shown that the lowest permeability of cell monolayer occurs between day 13 and day 15 after seeding45. Therefore, treatment of the THBMEC monolayer is performed after this time and bacteria are inoculated afterwards in the medium on the apical surface of the monolayer. After an incubation time, bacteria that were able to cross the barrier are quantified via plating medium with the bacteria on agar plates and counting the colonies. An increased number of colonies correlates with higher bacterial traversal through the BBB. The TEER is about 70 &#937;&#183;cm2 46. However, it is not necessary to measure the TEER in the described method. Although it is a well-established value for the permeability of the BBB, it seems to have no impact on the traversal of bacteria through the BBB. Untreated cells serve as a control of tightness in our model. It has been shown in previous work that the cells are able to react to proinflammatory cytokines and express typical tight junction proteins47. This allows for compound screening and validation of a larger set of transporter substrates and receptors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar - Agar</td>
			<td>Carl Roth</td>
			<td>6494.3</td>
			<td>BioScience-Grade</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Systec</td>
			<td>VX-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria E.coli strain GM2163</td>
			<td>Fermentas Life Sciences, Lithuania</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer</td>
			<td>Eppendorf</td>
			<td>6131</td>
			<td></td>
		</tr>
		<tr>
			<td>Cells THBMEC</td>
			<td>Group of M. F. Stins</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Greiner Bio-One</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Universal 320</td>
			<td>Hettrichlab</td>
			<td>1401</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>SIGMA Aldrich</td>
			<td>C6745</td>
			<td>from human cell culture</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>C10311</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>Greiner Bio-One</td>
			<td>191180</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>BRAND</td>
			<td>759015</td>
			<td></td>
		</tr>
		<tr>
			<td>Di sodium hydrogen phosphate di hydrate</td>
			<td>MERCK</td>
			<td>1065800500</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>GIBCO/ Thermo Sc.</td>
			<td>11330032</td>
			<td>HEPES</td>
		</tr>
		<tr>
			<td>Falcon tubes 15 ml</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 50 ml</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO/ Thermo Sc.</td>
			<td>10270</td>
			<td>value FBS -Brazil</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>SIGMA Aldrich</td>
			<td>F0556</td>
			<td>solution human fibroblasts</td>
		</tr>
		<tr>
			<td>Heracell 150 CO2 Incubator</td>
			<td>Heraeus</td>
			<td>50116047</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker I 26 New Brunswick</td>
			<td>Eppendorf</td>
			<td>M1324-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Dr. Ilona Schubert - Laborfachhandel</td>
			<td>641000</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth Base</td>
			<td>GIBCO/ Thermo Sc.</td>
			<td>12780029</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>GIBCO/ Thermo Sc.</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbial incubator B 6200</td>
			<td>Heraeus</td>
			<td>51015192</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological Safety Cabinet AURA 2000 M.A.C. Class II</td>
			<td>BIOAIR</td>
			<td>12469</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope inverse</td>
			<td>Zeiss</td>
			<td>TELAVAL 31</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tubes 2 ml</td>
			<td>Sarstedt</td>
			<td>72,695,400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tubes 1,5 ml</td>
			<td>Sarstedt</td>
			<td>72,706,400</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin</td>
			<td>GIBCO/ Thermo Sc.</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Dr. Ilona Schubert - Laborfachhandel</td>
			<td>464-800</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Roth</td>
			<td>HN02.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium-di-hydrogen phosphate</td>
			<td>Roth</td>
			<td>P018.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinCerts + Multiwell Plates</td>
			<td>Greiner Bio-One</td>
			<td>665631</td>
			<td>12 well, pore size 3.0 &#181;m</td>
		</tr>
		<tr>
			<td>Trypsin - EDTA</td>
			<td>GIBCO/ Thermo Sc.</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuumpump Laboport</td>
			<td>KNF</td>
			<td>N 86 KT.18</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the permeability of the blood-brain barrier by microbial traversal through microvascular endothelial cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

Following the protocol, the cells were seeded, and the BBB model was built. At day 14 after seeding, the cells were treated with glyoxal as a reactive aldehyde. The aim of the experiment was to investigate the correlation between age and diabetes in POD27 and the high incidence of meningitis in elderly patients48. The increased levels of advanced glycation end products (AGEs) in both age and diabetes49 demand further examination of the effect of glycation in the pathogenesis of microbial traversal through the BBB. Glycation is a non-enzymatic reaction of free amino groups in proteins with carbonyl groups of reducing carbohydrates or other carbonyl compounds. Glucose is well known as donor of carbonyl groups; however, there are more reactive ones known. After building an unstable Schiff&#39;s base, they rearrange to more stable and reactive dicarbonyl compounds like glyoxal. AGEs, the final products, can cause crosslinks between proteins50. They can damage cellular structures and alter cellular function by interaction with the receptor of AGEs (RAGE)51.
Cells were treated with a 0.05 and 0.15 mM glyoxal (GO) solution for 1 h, and untreated cells served as a control. Glycation was detected via immunoblotting and detection with an anti-AGE antibody (Figure 3). The obtained bacterial colonies were counted and represented as the absolute number of colonies (Figure 4A) or the relative number of colonies normalized to the control (Figure 4B). The medium taken from wells with the untreated cells formed very few colonies. This result demonstrated that the untreated cells were able to build a barrier and could serve as a control. Samples treated with glyoxal displayed an increased number of colonies, leading to the conclusion that there is an effect of glyoxal on the THBMECs and the cellular barrier density, because the number of colonies demonstrated a significant difference between untreated and treated cells. The increased bacterial crossing of the barrier after the treatment with glyoxal could explain why diabetes is correlated to diseases with a BBB breakdown.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60692/60692fig3a.jpg" /> 
Figure 3: Detection of protein glycation via immunoblotting. THBMECs were treated with GO at different concentrations for 1 h. Total protein was isolated and separated using SDS-PAGE. Glycation of the proteins was detected via immunoblotting using anti-AGE-antibody (CML-26). <a href="https://www.jove.com/files/ftp_upload/60692/60692fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In a different setting, protein glycation of THBMECs was induced by glucose. Sterilized glucose was added to the DMEM/F-12 medium to increase glucose concentration from normal glucose medium (NG) with 17.5 mM to high glucose medium (HG) with 42.5 mM. THBMECs were cultivated in two different cell culture flasks: one in normal glucose (NG) medium and the other in high glucose (HG) medium. These two different media were also used for growth of the BBB on filters in 12 well plates. Cells cultivated in NG medium served as a control. The obtained colonies are represented as the absolute number of colonies (Figure 4C) or the relative number of colonies normalized to the control (Figure 4D). The results indicate no significant effect on the traversal of bacteria through the human BBB, leading to the conclusion that the effect of NG vs. HG was not severe enough to affect the integrity of the BBB. The different scenarios were designed to prove the model and the integrity of the cells mimicking the BBB.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60692/60692fig4d.jpg" /> 
Figure 4: Absolute and relative numbers of counted bacterial colonies in the BBB model with THBMECs. THBMECs were treated with 0.15 mM GO for 1 h, untreated cells served as a control. A total of 450 &#181;L of E. coli suspension (OD600 = 0.5) was added to each upper chamber. Medium from the lower chambers was plated on agar plates after 6 h. (A) The graph shows the average mean +/- SEM of counted colonies. (B) The graph shows the counted colonies normalized to the untreated cells as control +/- SEM (n = 4). In (C) and (D), THBMECs were cultivated in NG and HG medium. A total of 450 &#181;L of E. coli suspension (OD600 = 0.5) was added to each upper chamber. Medium from the lower chamber was plated on agar plates after 6 h. (C) The graph shows the average mean +/- SEM of counted colonies. (D) The graph shows the counted colonies normalized to the untreated cells as control +/- SEM (n = 3). <a href="https://www.jove.com/files/ftp_upload/60692/60692fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

analyzing-permeability-blood-brain-barrier-microbial-traversal

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JoVE Journal

Immunology and Infection

15.0K Views.  Martin Luther University Halle-Wittenberg. The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

Video: Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

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.

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.

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

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...