an assay to measure chemically-induced cytotoxicity in human precision-cut lung slices

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices

To prepare a master mix of the working solution, dilute 25 microliters of the WST-1 reagent and 225 microliters of medium for each well. Next, pipette 250 microliters of the master-mix working solution of WST-1 for each well, and incubate the plate at 37 degrees Celsius for one hour. Ensure that the PCLS are fully covered by the WST-1 reagent during incubation.
Afterward, place the plate on an orbital shaker at 200 rpm, and shake carefully for 30 seconds to ensure thorough mixing of the WST-1 reagent. Then, pipette 100 microliters of the supernatant from each well of the 24-well plate to a new flat bottom 96-well plate in duplicates. Measure the absorption of each well at 450 nanometers using a microplate reader, and subtract the absorption at 630 nanometers reference from that at 450 nanometers.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. General Preparation of Human precision-cut lung slices (PCLS) and Subsequent Exposure to Chemicals
NOTE: Two persons are required to fill a lung. An up-to-date vaccination record for hepatitis A and B is recommended. Patients are routinely screened for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) prior to lung transplantation. If an active infection with&#160;Mycobacterium tuberculosis&#160;is diagnosed or suspected, the lung should be rejected. Nevertheless, all fresh human lung tissue and samples derived from it must be treated as potentially infectious, and corresponding protective measures must be taken (particle filter masks (FFP2), protective eyewear, gloves) to ensure the occupational safety of the staff. The procedure takes 60 - 90 min.
<ol>
	<li>Confirm the intactness of the human lung lobe. 
	NOTE: A tear in the pleura prevents homogeneous filling of the tissue. Human lung material must be from the day of resection. Storage periods of about 2 h at room temperature (RT) prior to filling it with agarose on ice are tolerated. The tissue that we use in this protocol is obtained from patients undergoing resection. It is not from deceased patients. If the tissue has been stored on ice, pre-warm it to RT before filling it, otherwise, the agarose will polymerize immediately, and no homogeneous filling will be possible.&#160;&#160;&#160;&#160;&#160; 
	CAUTION: Ensure that all persons in contact with native human material put on protective clothing consisting of a lab coat, two pairs of gloves, a cap, a face mask, and a pair of safety glasses. Human material is potentially infectious.</li>
	<li>Weigh 7.5 g of low-gelling agarose and add it to 250 mL of bi-distilled water. Boil the agarose in a microwave until the agarose is dissolved. Cool it to approximately 40 &#176;C, depending on the melting and gelling point of the agarose. 
	NOTE: Several flasks are required, depending on the lobe size.</li>
	<li>Pre-warm and keep the culture medium (Table of Materials) at 37 &#176;C. 
	NOTE: If the agarose is too hot, vitamins in the culture medium will lose effectiveness and cells will be damaged. If the temperature of the medium/agarose solution is below 37 &#176;C, the gelling process will start and impair the homogeneous filling of the lung. Only a temperature range of 37 &#176;C to 39 &#176;C is recommended.</li>
	<li>Place all the required materials within reach before starting: 5 - 10 clamps, a flexible catheter of approximately 1 m length, a suitable syringe (e.g., 50 mL) fitting the connection to the catheter, and an ice box filled with ice.</li>
	<li>Cannulate the trachea/main bronchus by inserting the silicone tube and fixating it with a clamp oriented parallel to the tube, so that the clamp squeezes the tissue together alongside the silicone tube without pinching it off. Verify that the silicone tube is fixated inside and cannot slip out during the filling procedure. Close all other bronchi, blood vessels, and injuries with clamps, so that no agarose can leak out during the filling procedure.</li>
	<li>Mix an equivalent volume of 3% low-gelling agarose with culture medium in a beaker. Instill the mixture into the lung using a 50-mL syringe. Prior to refilling the syringe with medium, clamp shut the catheter with fingers or a clamp to avoid air bubbles and agarose reflux. Fill the lung lobe until it is fully inflated. Carefully touch the lung pleura on the side; the pleura should be even and hard. 
	NOTE: Depending on the size of the lung lobe, up to 2 or 3 L of agarose/medium solution can be required.</li>
	<li>Repeat steps 1.5 - 1.6 for each bronchus in the case of several bronchi within a single specimen.</li>
	<li>Put the lung on ice for polymerization of the agarose to gel for 20 - 40 min, depending on the amount of instilled agarose. Carefully touch the pleura on several sides to check whether it is hard and cool, indicating that polymerization is complete, otherwise, continue to wait. The pathologists will cut the lung into slices, take their samples, and store the lung material on ice for transportation.</li>
	<li>Cut the human lung tissue into 3 - 5 cm slabs with a sharp knife.&#160;&#160;&#160;&#160; 
	NOTE: Use a new blade for every lung to ensure sharpness.</li>
	<li>Fill the tissue slicer with 400 mL ice-cold Earle&#39;s balanced salt solution (EBSS). Immediately cut cylindrical tissue cores out of the lung slabs using a semi-automated screwdriver with a coring tool of the preferred diameter (e.g., 8 mm; 10 mm). 
	NOTE: This diameter must be equivalent to the diameter of the tissue holder inside the machine.</li>
	<li>Adjust the thickness of the lung slices to the desired thickness value. 
	NOTE: A thickness of approximately 250 &#181;m is commonly used in PCLS experiments. The manufacturer of the tissue slicer recommends their Tissue Slice Thickness Gauge for verification of the slice thickness. Alternatively, whole-mount staining combined with confocal microscopy can be used to determine the slice thickness as described by Brismar&#160;et al.</li>
	<li>Transfer the tissue cores into the tissue holder of the tissue slicer. Put the weight (part of the tissue holder) on top of the tissue core. Set the arm speed and blade speed to 6 on the tissue slicer. Start slicing the tissue core into PCLS.</li>
	<li>Supplement 500 mL of commercially available Dulbecco&#39;s Modified Eagle Medium (DMEM): Nutrient Mixture F-12 DMEM/F12 (1:1) with 5 mL of penicillin (10,000 units/mL) and streptomycin (10,000 &#181;g/mL). Store the culture medium for several days under sterile conditions in a refrigerator. Pre-warm only the required volume of the medium.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Penicillin will be inactivated if kept at 37 &#176;C for longer periods. Use serum-free and dye-free culture medium, as serum composition varies from batch to batch, and dyes, such as phenol-red, can interfere with assays.</li>
	<li>Fill a Petri dish (100 x 15 mm) with 25 mL ice-cold culture medium. The medium should be drained out of the tissue slicer into a beaker by opening the clamp of the glass cylinder. Transfer the slices from a beaker into the Petri dish with culture medium by using an applicator (e.g., inoculation loop). Put the Petri dish into an incubator (37 &#176;C, 5% CO2, 100% humidity). Allow the medium to warm up prior to washing steps. 
	NOTE: All further steps are performed under sterile conditions.</li>
	<li>Place a cell strainer into the Petri dish for washing the PCLS. Completely remove the culture medium with a 10-mL serological pipette through the cell strainer and add 25 mL of 37 &#176;C pre-warmed fresh medium. Repeat this step 3 - 4 times every 30 min.&#160;&#160;&#160;&#160;&#160; 
	NOTE: The cell strainer prevents the slices from being sucked into the pipette, to avoid any damage to the slices.</li>
	<li>Transfer the lung slices carefully into a 24-well culture plate with a minimum of 500 &#181;L culture medium for two slices per well. Expose the lung tissue to substances (see steps 3.1 - 3.4),&#160;e.g., for 24 h at 37 &#176;C, 5% CO2.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: For the transfer, preferably use an inoculation loop and let the slices float onto the loop in order to prevent tissue damage. No further separation of the slices is needed, as only a shortage of sufficient fresh medium will induce cell death in tissue slices. Slices can overlap or touch each other in the well: this has no influence on tissue viability.</li>
	<li>Put all residual human material into a plastic flask with a fixative (e.g., 10% buffered formaldehyde) for at least 24 hours and burn this in the disposal process. 
	CAUTION: Formaldehyde is toxic, perform this step under a hood.</li>
</ol>
2. Preparation of Solutions for Substances
NOTE: Prepare working solutions and controls immediately before use.
CAUTION: Handle substances according to safety instructions or, if unknown, as potentially harmful and follow routine safety precautions.
<ol>
	<li>Dissolve water-soluble substances directly in the culture medium. For insoluble or poorly soluble chemicals, first dissolve the substance in the appropriate solvents depending on substance solubility. Substances with limited water solubility (&#60;0.1 mg/mL) can be dissolved, for example, in dimethyl sulfoxide (DMSO) or ethanol. Non-toxic solvent concentrations should be determined by titration beforehand. Make sure the substances do not precipitate out of the solution when diluted into the medium. 
	NOTE: ammonium hexachloroplatinate (HClPt) and sodium laureth sulfate (SLS) solutions are prepared.</li>
	<li>Prepare substance stock solutions at 100-fold of the desired highest concentration in the culture medium or solvent. Weigh 12.5 mg HClPt and 34.4 mg SLS and prepare stock solutions by dissolving the chemicals in 1 mL culture medium. 
	NOTE: No solvent is required for these chemicals.</li>
	<li>Prepare a final dilution of 1:100 in a pre-warmed medium. In the case of prior use of solvent, this approach results in the same final solvent concentration for all substance concentrations.</li>
	<li>Use the final solvent concentration (e.g., 1% as described in step 3.3) for reference treatment of PCLS. No solvent control was required for the chemicals mentioned in the results section.</li>
</ol>
3. Positive and Negative References for Cytotoxicity Assays
<ol>
	<li>For all viability assays, prepare the following positive and negative controls:
	<ol>
		<li>Tissue control: incubate PCLS in a culture medium only as a reference for untreated PCLS for 24 h at cell culture conditions (37 &#176;C, 5% CO2, 100% humidity).</li>
		<li>Vehicle control (if necessary): incubate PCLS with the final solvent concentration as a reference for PCLS treated with vehicle only (see step 3.4) for 24 h at cell culture conditions (37 &#176;C, 5% CO2, 100% humidity).</li>
		<li>Positive control: incubate PCLS with 1% detergent in buffer solution for 1 h at 4 &#176;C.&#160;&#160;&#160;&#160;&#160; 
		NOTE: If PCLS becomes colorless, the tissue is dead. Total L-lactate dehydrogenase (LDH) is determined in the supernatant, with an absorption of approximately 1.9 - 2.3. The tissue is used for the WST-1 assay, with an absorption of approximately 0.</li>
	</ol>
	</li>
</ol>
4. Measurement of Chemically-induced Cytotoxicity in Human PCLS by water-soluble tetrazolium salt (WST-1) Assay
NOTE: The WST-1 assay is performed in a 24-well plate with two PCLS per well. Preferably, use duplicates for each parameter and pool the results of these duplicates after measurement.
<ol>
	<li>Prepare the working solution by diluting the WST-1 reagent in the medium immediately before starting. The required amount of working solution is 250 &#181;L/well of a 24-well plate. Therefore, mix 25 &#181;L of the reagent with 225 &#181;L of the culture medium for one well.</li>
	<li>After incubation of PCLS from step 1.16, discard or use the supernatant for cytokine measurements or other tests such as the LDH assay. The remaining tissue is used for the next step.</li>
	<li>Pipet 250 &#181;L of the working concentration of the WST-1 reagent per well and incubate the plate at 37 &#176;C and 5% CO2&#160;for 1 h. Ensure that the PCLS are fully covered by the WST-1 reagent during incubation.</li>
	<li>Place the plate on an orbital shaker (200 rpm) and shake carefully for 30 s to ensure thorough mixing of the WST-1 reagent. Take a new flat-bottom 96-well plate and pipet 100 &#181;L in duplicates from the supernatant of each well of the 24-well plate.</li>
	<li>Measure the absorption of each well at 450 nm (reference: 630 nm) using a microplate reader. Subtract the absorption at 630 nm from 450 nm. NOTE: Reliable data for cytotoxicity assessment can be obtained only from viable tissue. Therefore, these acceptance criteria published should be met in each experiment. If these criteria are not met, the experiment should be repeated.</li>
	<li>Absorption of the untreated medium control should be above 0.6, otherwise, the experiment should be repeated. If the data meet this requirement, set the absorption value of the tissue control to 100% and calculate the WST-1 reduction of treated samples in relation to the tissue control.</li>
</ol>

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		<tr>
			<td>Silicon hose 3.0 x 5.0 mm</td>
			<td>A. Hartenstein (Leipzig, Germany)</td>
			<td>SS04</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Faust Lab Science (Klettgau, Germany)</td>
			<td>9.410 050</td>
			<td></td>
		</tr>
		<tr>
			<td>Coring tools</td>
			<td>custom-made</td>
			<td>custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimming Blade Handle (FEATHER)</td>
			<td>pfm medical ag&#160; (Cologne, Germany)</td>
			<td>205530001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimming Blades (FEATHER)</td>
			<td>pfm medical ag&#160; (Cologne, Germany)</td>
			<td>205500000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome: Tissue Slicer</td>
			<td>Alabama R&#38;D (Bad Homburg, Germany)</td>
			<td>303400-ADPT</td>
			<td>Krumdieck Tissue Slicer (MD6000)</td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Wilkinson Sword (Solingen, Germany)</td>
			<td>ENR-4027800011506</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer filter (100 &#181;m Nylon)</td>
			<td>Becton Dickinson (Heidelberg, Germany)</td>
			<td>BD352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Copan Diagnostics (Murrieta, USA)</td>
			<td>&#160;CD176S01</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue culture plates 24wells</td>
			<td>Sigma (M&#252;nchen, Germany)</td>
			<td>Z707791-126EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MaxiSorp flat-bottom</td>
			<td>Fisher Scientific GmbH (Hannover, Germany)</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well&#160;</td>
			<td>Thermo Scientific (Schwerte, Germany)</td>
			<td>260836</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell-Reader</td>
			<td>Tecan Group Ltd. (M&#228;nnedorf, Switzerland)</td>
			<td>Tecan infinite F200Pro</td>
			<td>Plate Reader</td>
		</tr>
		<tr>
			<td>Plate shaker</td>
			<td>Edmund Buehler GmbH (Hechingen, Germany)</td>
			<td>KM-2 Akku</td>
			<td></td>
		</tr>
		<tr>
			<td>Assays</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Proliferation Reagent WST-1</td>
			<td>Roche (Basel, Switzerland)</td>
			<td>11644807001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, low gelling temperature&#160;</td>
			<td>Sigma (M&#252;nchen, Germany)</td>
			<td>A9414-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Balanced Salt Mixtures and Solutions, Cell Culture, Classic Media and Salts, Earle&#39;s Balanced Salts (EBSS)</td>
			<td>Sigma (M&#252;nchen, Germany)</td>
			<td>E2888-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Lonza (Verviers, Belgium)</td>
			<td>17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle&#180;s Medium Nutrient Mixture F-12 Ham (DMEM F-12)</td>
			<td>Gibco (Darmstadt, Germany)</td>
			<td>11039-047</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Phosphate with Ca and Mg (DPBS)</td>
			<td>Lonza (Verviers, Belgium)</td>
			<td>BE17-513F&#160;&#160;</td>
			<td>Buffer solution</td>
		</tr>
		<tr>
			<td>Detergent</td>
			<td>Sigma (Saint Louis, USA)</td>
			<td>X100-100ML</td>
			<td>Triton X-100</td>
		</tr>
	</tbody>
</table>

Source: Neuhaus, V. et al., <a href="https://app.jove.com/v/57042">Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.</a>&#160;J. Vis. Exp.&#160;(2018)
This video illustrates a technique for evaluating cytotoxicity in human precision-cut lung slices through the WST-1 assay. Human precision-cut lung slices are subjected to incremental concentrations of a cytotoxic chemical to quantify their cytotoxicity. A decrease in color intensity serves as confirmation of the chemical&#39;s detrimental impact on lung cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. General Preparation of Human ...

Take a multi-well plate containing human precision-cut lung slices, which maintain the native lung microarchitecture.
Remove media. Add increasing test agent concentrations to the wells and incubate.
Depending on its cytotoxic potential, the test agent disrupts cell membranes, inducing cell death and reducing viable cells within the slices.
Post-incubation, remove media. Immerse the slices in media containing water-soluble tetrazolium salt, or WST-1, and an electron-coupling reagent.
Incubate. In metabolically active cells, the plasma membrane electron transport system transfers electrons from intracellular NADH to the extracellular electron-coupling reagent, reducing the cell-impermeable dye WST-1 to stable, dark-yellow, water-soluble formazan.
Post-incubation, shake the plate to evenly distribute the formazan and transfer it to a multi-well plate.
Using a microplate reader, measure the absorbance of formazan, indicative of the tissue viability.
A decrease in formazan absorbance, dependent on the test agent concentration, indicates reduced lung slice viability, confirming the cytotoxicity&#160; of the test agent.&#160;&#160;

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비디오: 인간의 정밀 절단 폐 절편에서 화학적으로 유도된 세포 독성을 측정하기 위한 분석 - 실험

Cytotoxicity Assays with Zebrafish Cell Lines

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

This protocol presents commonly used cytotoxicity assays (Alamar Blue [AB], CFDA-AM, Neutral Red, and MTT assays) adapted for the assessment of cytotoxicity in zebrafish embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates.

Zebrafish 세포주를 이용한 세포독성 분석

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute ...

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JoVE Journal

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An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

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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.

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.

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

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices

내레이션 대본

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices