This work was supported by the Ramalingaswami grant DBT-843-BIO (Department of Biotechnology, Government of India) and Early Career Research Award SER-1058-BIO (Science and Engineering Research Board, Government of India) to S.R. The authors acknowledge an ICMR-JRF grant to P.C and DBT-JRF grant to P.S. The authors thank Dr. Ravikant Ranjan for suggestions on the manuscript and technical assistance by Mr. Pradeep Singh Thakur during SEM.

BALB/c mice were housed in the Small Animal Facility at IIT Roorkee. All animals were maintained in a 12 h:12 h light:dark cycle at 25 &#176;C and were provided with a pellet diet and water ad libitum. All animal procedures were approved by the Institutional Animal Ethics Committee (IAEC) of IIT Roorkee.
1. Preparation of C. tropicalis
NOTE: The fungus Candida tropicalis belongs to Risk Group 2 pathogens and is classified as a BSL2 microorganism. Always use certified Class II Biological Safety Cabinets when working with Candida species. Practice aseptic and sterile techniques during work with C. tropicalis and follow the recommended biosafety procedures for proper disposal of this pathogen.
<ol>
	<li>Streak C. tropicalis (strain ATCC 750) onto a Sabouraud dextrose (SAB) agar plate.</li>
	<li>Prepare an overnight grown culture of C. tropicalis by inoculating a single colony from the SAB agar plate into a sterile 50 mL conical tube containing 10 mL of SAB broth medium. Alternatively, use a frozen glycerol stock of C. tropicalis and inoculate 100 &#181;L of the glycerol stock into a sterile 250 mL conical flask containing 50 mL of SAB broth medium.</li>
	<li>Incubate C. tropicalis culture in an orbital shaker at 180 rpm at 30 &#176;C for 24-48 h.</li>
	<li>Centrifuge the fungal culture (cells in logarithmic phase) at 2,150 &#215; g for 15 min at 21 &#176;C.</li>
	<li>Discard the supernatant and add 50 mL of sterile 1x PBS to the pellet. Wash and resuspend the pellet in sterile 1x PBS with gentle vortexing.</li>
	<li>Centrifuge again at 2,150 &#215; g for 15 min at 21 &#176;C. Discard the supernatant and resuspend the fungal pellet in 10 mL of sterile 1x PBS.</li>
	<li>Calculate the concentration of cells by counting with a haemocytometer.</li>
	<li>Prepare fungal stocks at a final density of 1.0 &#215; 106 cells/mL in RPMI 1640 morpholinepropanesulfonic acid (MOPS) medium. Use the cell suspension from step 1.6 immediately. 
	&#8203;NOTE: To set up one 96-well plate, the total fungal stock volume needed is 10 mL (100 &#181;L/well). Scale as needed.</li>
</ol>
2. C. tropicalis biofilm formation
<ol>
	<li>Prepare Candida biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier (Figure 1)28,29.</li>
	<li>Add 100 &#181;L of C. tropicalis culture (from 106 cells/mL stock, prepared as above) to a 96-well microtiter plate using a multichannel pipette (Figure 2A). Keep the last two columns (11 and 12) as &#39;no fungus plus serum&#39; and &#39;no fungus and no serum&#39; negative controls by not adding fungal cells. Fill columns 11 and 12 with 100 &#181;L of RPMI 1640 MOPS medium alone.</li>
	<li>Cover the microtiter plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 &#176;C under stationary conditions.</li>
	<li>The next day, aspirate the medium carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual medium.</li>
	<li>Wash the plate with 200 &#181;L of 1x PBS (per well) using a multichannel pipette. Add PBS very gently along the side walls of the well to avoid disrupting biofilms. Aspirate the PBS carefully using a multichannel pipette. Repeat the PBS wash 2x (a total of three washes).</li>
	<li>To remove excess PBS, air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet.</li>
</ol>
3. Treatment of biofilm with antibodies
NOTE: Biofilms can now be processed for assessing the inhibition of biofilm maturation by antibodies. Murine serum was used as the source of polyclonal antibodies. Different groups of Sap2-immunized (Sap2-albicans, Sap2-tropicalis, and Sap2-parapsilosis) along with sham-immunized mice were bled retro-orbitally and serum was isolated as described earlier19. The presence of anti-Sap2 antibodies was confirmed using Sap2-specific ELISA as described previously19.
<ol>
	<li>Perform heat-inactivation of the serum (source of polyclonal antibodies) at 56 &#176;C for 30 min before use to rule out the role of complement in the inhibitory activity. Heat-inactivate the serum before making serum dilution. 
	NOTE: Use serum from sham-immunized mice, preimmune mice, and Sap2-specific antibody-depleted serum as additional controls19. Antibody-depleted serum was prepared as per a previous study19. Among the serial dilutions (1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, and 1:12,800) for serum tested in this protocol, inhibition of biofilm maturation was observed at 1:25, 1:50, and 1:100; hence, 1:50 was selected to strike a balance between inhibition and serum consumption.</li>
	<li>Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI 1640 MOPS medium (1:50). Use a common serum dilution (1:50) for all the serum samples to be tested for inhibition of biofilm maturation30.</li>
	<li>Add 100 &#181;L of the selected serum dilution to each well of the 96-well microtiter plate. For each sample, add serum dilutions in duplicate, as per the layout attached (Figure 2B).
	<ol>
		<li>In column 10, do not add serum dilution; add only RPMI 1640 MOPS medium for the fungus-only positive control. 
		NOTE: Column 10, rows G1-G8 and H1-H8 initially had fungal cells in RPMI-MOPS. However, while RPMI-MOPS was added to column 10 even after 24 h, rows G1-G8 served as PBS control and rows H1-H8 as no-serum control after 24 h.</li>
		<li>In column 11, add a 1:50 dilution of serum to all wells to serve as the no fungus plus serum negative control.</li>
		<li>In column 12, do not add serum dilution to any well; keep this as the no fungus no serum negative control.</li>
	</ol>
	</li>
	<li>Cover the plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 &#176;C.</li>
</ol>
4. Biofilm metabolic activity estimation
<ol>
	<li>The next day, aspirate the serum carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual serum.</li>
	<li>Wash the plate with 200 &#181;L of 1x PBS (per well) using a multichannel pipette, adding the PBS along the side walls of the well to avoid disrupting the biofilms. Aspirate the PBS carefully using a multichannel pipette and repeat the PBS wash 2x (a total of three washes). Air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet to dry any excess PBS.</li>
	<li>Preparation of XTT/menadione:
	<ol>
		<li>Prepare XTT in sterile Ringers Lactate as a 0.5 g/L solution. Dissolve 25 mg of XTT in 50 mL of filter-sterilized Ringers Lactate. Aliquot 10 mL in separate tubes covered with aluminum foil and store at -80 &#176;C.</li>
		<li>Prepare menadione as a 10 mM stock. Dissolve 8.6 mg of menadione in 5 mL of acetone and distribute 50 &#181;L in 100 separate microtubes. Store the aliquots at -80 &#176;C.</li>
		<li>Prepare XTT/menadione solution just before use by taking 10 mL of XTT and adding 1 &#181;L of menadione to obtain a 1 &#181;M working solution.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of the XTT/menadione solution per well of the 96-well microtiter plate. Cover the plate with a lid and aluminum foil. Incubate the plate for 2 h at 37 &#176;C in the dark.</li>
	<li>Transfer 80 &#181;L of the colored supernatant from each well into a fresh 96-well plate. Read the plate at 490 nm.</li>
	<li>Calculate the mean of the absorbance values of the wells in column 10 (fungus-only positive control), which will serve as a reference value for calculating the percentage biofilm inhibition by each serum sample using equation (1). 
	% Biofilm inhibition = 100 - <img alt="Equation 1" src="/files/ftp_upload/64425/64425eq01.jpg" style="margin: auto;" /> &#215; 100&#160; &#160;&#160;(1)</li>
</ol>

The authors declare no conflicts of interest.

Fungal infections&#160;caused by Candida species are associated with high morbidity and mortality rates worldwide. The growing threat of invasive fungal infection requires the early management of such life-threatening diseases. Most Candida infections involve the formation of biofilms, which adhere to a variety of medical devices and are responsible for the persistence and recurrence of fungal infections in hospital settings31. Biofilms are composed of yeast or hyphal cells, and ...

Systemic candidiasis is the fourth major cause of nosocomial infections, which are associated with high morbidity and mortality rates worldwide. Globally, systemic candidiasis affects approximately 700,000 individuals1. Candida species, namely C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. auris, are the most common cause of invasive Candida infections2. Candida species are opportunistic pathogens that produce biofilms3. Biofilms are predominantly associated with Candida virulence, and Candida can withstand oxidative and osmotic stress conditions by inducing biofilm formation4. Biofilms further modulate the expression of virulence factors and cell wall components and form an exopolymeric protective matrix, helping Candida to adapt to different host niches4. Biofilms contribute to yeast adherence on host tissues and medical instruments5. As such, biofilm formation is associated with an advantage to yeasts, as yeast cells within the biofilms can evade the host immune response6. Biofilm formation also protects the pathogenic yeasts from the action of antifungal drugs5. Decreased susceptibility of C. albicans biofilms to amphotericin B has been demonstrated by Pierce et al.7,8. Furthermore, biofilms demonstrate antifungal drug resistance to fluconazole, which impairs effective management of systemic candidiasis9,10.
Microbes have an intrinsic tendency to adhere to various biotic and abiotic surfaces, which results in biofilm formation. Candida albicans, which is a dimorphic fungus, exists in yeast and hyphal forms,&#160;and its biofilm formation has been characterized in various in vitro and in vivo model systems11. The steps of biofilm formation include the adhesion of Candida cells to the substrate, filamentation, proliferation, and biofilm maturation11. Initially, the yeast form of C. albicans adheres to substrates, including medical devices and human tissue, followed by filamentation and proliferation of C. albicans into hyphal and pseudohyphal forms, and finally maturation of biofilms embedded in extracellular matrix11. Biofilm formation largely contributes to C. albicans pathogenesis mechanisms12. Candida species form drug-resistant biofilms, which makes their eradication challenging13. A small subset of the C. albicans biofilm-producing population has been described as being highly resistant to the antifungal drugs amphotericin B and chlorhexidine14. Of note, yeast cells in biofilms exhibit high resistance to multidrug therapy compared to yeast cells in the planktonic phase and proliferation phase14. It has been suggested that yeast cells existing in biofilms are highly tolerant to antifungal drugs, which contributes to C. albicans survival in biofilms14. These existing cells were reported to be phenotypic variants of C. albicans and not mutants14. Furthermore, cells of Candida biofilms known as &#34;persister cells&#34; are tolerant to high doses of amphotericin-B treatment and contribute to Candida survival, thereby posing a great burden of recurring systemic Candida infections in high-risk individuals15.
The increase in antifungal drug resistance in Candida strains necessitates research for new antifungal agents and immunotherapies. As evident from the abovementioned studies, Candida biofilms show decreased susceptibility to antifungal drugs. Therefore, there is a need for improved immunotherapies to control Candida biofilm formation. Earlier studies have shown that CAGTA can provide effective protection against systemic Candida infections by inhibiting C. albicans biofilm formation in vitro16. Another study reported that immunization of mice with C. albicans rAls3-N protein induces high antibody titers that interfere with C. albicans biofilm formation in vitro17. Anti-Als3-N&#160;antibodies also exerted an inhibitory effect on C. albicans dispersal from biofilms17. NDV-3A vaccine based on C. albicans is currently under clinical trial and anti-NDV-3A sera were also found to reduce C. auris biofilm formation18. A recent study identified inhibition of biofilm formation by Sap2-antibodies as a protection mechanism in a murine model of systemic candidiasis19.
This paper outlines a detailed in vitro protocol for evaluating the effect of antigen-specific antibodies present in polyclonal serum obtained from different groups of Sap2 vaccinated mice on preformed Candida tropicalis biofilms. To achieve this, a method based on an XTT reduction assay was optimized and developed in the laboratory, which can measure biofilm viability in a fast, sensitive, and high-throughput manner, in the presence or absence of antibodies.
The XTT assay is used to measure cellular metabolic activity as an indicator of cell viability, cellular proliferation, and cytotoxicity20. This colorimetric assay is based on the reduction of a yellow tetrazolium salt, sodium 3&#180;-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) to an orange formazan dye by metabolically active cells. Since only viable cells can reduce XTT, the amount of reduced XTT formazan is proportional to the intensity of color and cell viability. The formazan dye formed is water-soluble and is directly quantified using a plate reader. Due to its water-soluble nature, the XTT assay allows the study of intact biofilms, as well as the examination of biofilm drug susceptibility, without disruption of biofilm structure21. Additionally, this method is implemented in Candida fungal viability assessments due to its ease of use, speed, accuracy, high throughput, and high degree of reproducibility7,22.
In addition to the XTT reduction assay, numerous alternative techniques have also been identified for the measurement of biofilm quantity. Some of these include the use of the MTT reduction assay, crystal violet staining, DNA quantification, quantitative PCR, protein quantification, dry cell weight measurement, and viable colony counting. These procedures vary widely in terms of their time and cost requirements. Taff et al. performed a comparative analysis of seven different Candida biofilm quantitation assays and found that the XTT assay provided the most reproducible, accurate, and efficient method for the quantitative estimation of C. albicans biofilms23. Staining techniques such as crystal violet have certain limitations; the crystal violet test indirectly determines the amount of biofilm by measuring the optical density of the crystal violet-stained biofilm matrix and cells. Although the crystal violet assay provides a good measure of biofilm mass, it does not give a measure of biofilm viability as it stains both microbial cells and the extracellular matrix24. Dhale et al. further reported that the XTT reduction assay was the most sensitive, reproducible, accurate, efficient, and specific method to detect biofilm production as compared to crystal violet assay25. Literature reports have shown that the XTT assay correlates well with the CFU/mL parameter in the CFU counting method. However, compared to the XTT assay, the CFU method is labor-intensive and slow26. Furthermore, the fraction of detached live cells may not be representative of the initial biofilm population27. Although the XTT reduction assay seems the best available option to quantify viability, there are a few limitations of this technique. While the XTT method is useful for comparisons involving one fungal strain, its use may be limited when comparing different fungal strains and species. Interstrain comparisons may be difficult in the absence of detailed standardization since different strains metabolize substrates with different capabilities21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>-</td>
			<td>Prepared in lab</td>
			<td>NaCl : 4 g 
			KCl : 0.1 g 
			Na2HPO4:&#160; 0.72 g 
			KH2PO4 : 0.12 g 
			Water 500 mL. Adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtiter plates</td>
			<td>Nunc</td>
			<td>442404</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Menadione</td>
			<td>Sigma</td>
			<td>M5625</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter Plate Reader</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette and tips</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Tarson</td>
			<td>460090</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringers Lactate</td>
			<td>-</td>
			<td>Prepared in lab</td>
			<td>sodium chloride 0.6 g sodium lactate 0.312 g potassium chloride 0.035 g calcium chloride 0.027 g Water 100 mL. Adjust to pH 7.0&#160;</td>
		</tr>
		<tr>
			<td>RPMI 1640 MOPS</td>
			<td>Himedia</td>
			<td>AT180</td>
			<td></td>
		</tr>
		<tr>
			<td>Sabouraud dextrose Agar</td>
			<td>SRL</td>
			<td>24613</td>
			<td></td>
		</tr>
		<tr>
			<td>Sabouraud dextrose Broth</td>
			<td>SRL</td>
			<td>24835</td>
			<td></td>
		</tr>
		<tr>
			<td>XTT&#160;</td>
			<td>Invitrogen</td>
			<td>X6493</td>
			<td></td>
		</tr>
	</tbody>
</table>

a soluble tetrazolium-based reduction assay to evaluate the effect of antibodies on candida tropicalis biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Candida tropicalis biofilms were grown in 96-well microtiter plates and imaged at 40x using an inverted microscope (Figure 1A). The biofilm was further stained using crystal violet and observed at 40x using an inverted microscope (Figure 1B). Scanning electron microscopy shows a representative image of C. tropicalis biofilm (Figure 1C). For performing the biofilm inhibition assay, 105 cells of Candid...

Watch this Scientific Journal Video about A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms at JoVE.com

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

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Immunology and Infection

3.0K Views.  Indian Institute of Technology Roorkee. A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Video: A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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

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

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

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

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.