The authors would like to thank members of the Keyel and Zhang labs for their critical review of the manuscript. The authors thank the College of Arts and Sciences Microscopy for the use of facilities.

All appropriate guidelines and standard microbiological, safety, and cell culture practices were employed for the use and handling of the RG2 pathogen Leishmania major and recombinant DNA. All experiments with live L. major were performed in a biosafety cabinet in a BSL-2 certified laboratory. The work was overseen by the Texas Tech University Institutional Biosafety Committee.
NOTE: From a safety perspective, live L. major promastigotes are Risk Group 2 pathogens. H...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESCRT+machinery+is+required+for+plasma+membrane+repair.">ESCRT machinery is required for plasma membrane repair.</a> Science. 343 (6174), 1247136 (2014).</li><li>Demonbreun, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+actin-dependent+annexin+complex+mediates+plasma+membrane+repair+in+muscle.">An actin-dependent annexin complex mediates plasma membrane repair in muscle.</a> Journal of Cell Biology. 213 (6), 705-718 (2016).</li><li>Wolfmeier, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2)(+)-dependent+repair+of+pneumolysin+pores:+A+new+paradigm+for+host+cellular+defense+against+bacterial+pore-forming+toxins.">Ca(2)(+)-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins.</a> Biochimica et Biophysica Acta. 1853 (2), 2045-2054 (2015).</li><li>Bravo, F., Sanchez, M. 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In this study, methods to study the molecular mechanisms and functions of PFTs were described, using the human pathogen Leishmania major as a model system. A medium-throughput flow cytometry-based cytotoxicity assay to measure single-cell viability was developed. Viability is quantitative at the population level because LC50 values can be calculated from the dose-response curve using logistic modeling. As a proof-of-principle, a flow cytometric assay was used to illustrate that the choice of media can...

Pore-forming toxins (PFTs) are the largest family of bacterial toxins1, but the mechanisms by which they perforate and destroy cells are poorly understood. The best-studied family of pore-forming toxins is that of cholesterol-dependent cytolysins (CDCs). CDCs are primarily synthesized by gram-positive bacteria, including the causative agent of necrotizing fasciitis, Streptococcus pyogenes2. S. pyogenes secretes the CDC streptolysin O (SLO), which binds to sterols in the plasma membrane of host cells as monomers, oligomerizes, and inserts ~20-30 nm pores into the membrane1. The role that lipids play in this process remains poorly determined.
One approach to studying lipid-CDC interactions is the use of chemically defined liposomes. While defined liposomes provide information on the necessary thresholds of lipids to sustain toxin binding and pore formation3,4, they do not fully recapitulate cellular functions. For example, reconstituted liposomes lack the lipid asymmetry of mammalian hosts and lipid modifications in response to toxins5. One alternative to liposomes is to use mammalian cell lines. While these cell lines are more physiologically relevant, there is a large degree of redundancy in toxin sensing and resistance mechanisms2. As a consequence, the repair pathways used to resist CDCs remain poorly determined. Notably, Ca2+ influx is the primary activator of membrane repair1. Downstream of Ca2+ influx, multiple pathways are engaged, including a ceramide-dependent repair6,7 and a MEK-dependent repair pathway6. These pathways interact with other protein effectors, including the endosomal sorting complex required for transport (ESCRT)8, and annexins6,9,10. Dissecting these pathways in mammalian cells is challenging due to the redundancy, which muddles data interpretation.
One way to balance complexity with simplicity for dissecting repair pathways is the use of simpler organisms, such as protozoan pathogens in the genus Leishmania. Leishmania sp. cause leishmaniasis in humans and other animals. Leishmaniasis ranges from cutaneous leishmaniasis (self-limited skin lesions) to the fatal visceral leishmaniasis (hepatosplenomegaly), depending on the species and other factors11. Leishmania major, the causative agent of cutaneous leishmaniasis, is transmitted to humans via a sandfly vector and is used to understand Leishmania function and infection12. In addition, Leishmania sp. are digenic12. They exist as intracellular mammalian macrophage parasites termed amastigotes and as free-swimming, flagellated promastigotes in the sandfly12.&#160;L. major promastigotes can be cultured in serum-supplemented media such as M199 to high density13. Promastigotes are also genetically tractable; many gene knockouts exist, including those targeting lipid biosynthesis pathways13. These knockouts can be evaluated for growth and differences in infectivity and lesion development via infection of Balb/c mice13.
In addition to the relative ease of Leishmania culture and the range of lipid biosynthesis knockouts, the parasite has a simpler genome than mammals. The best-characterized species of Leishmania is L. major, which has many existing genetic tools, such as mutants with defective lipid metabolism14. Notably, many repair proteins are absent. L. major has no homologs identified to date for key mammalian repair proteins such as annexins. This enables the characterization of evolutionarily conserved repair pathways without the complexity of mammalian systems. However, repair pathways have not been characterized in Leishmania to date. At the same time, key signaling pathways involved in repair, such as the MEK pathway6, are conserved in Leishmania sp.15,16, though homologs need to be validated. The mitogen-activated protein kinase (MAPK) pathway is well-studied in L. mexicana, where it contributes to intracellular survival and thermostability in mammalian cells and controls metacyclogenesis16. In Leishmania sp., 10 of the 15 MAPKs have been characterized17. LmMAPK9 and LmMAPK13 are predicted to be the most similar to mammalian ERK1/2 based on identity in the conserved phosphorylation lip sequence. The phosphorylation lip sequence is TEY for both mammalian ERK1/2 and LmMAPK9 and LmMAPK13. However, eight of the Leishmania MAPKs have a TDY phosphorylation motif15. At least two homologs of MEK have been identified in Leishmania sp., LmxMKK18 and MEKK-related kinase (MRK1)19. This suggests that insights identified in Leishmania could translate to mammalian systems. Where they do not translate to mammalian systems, they represent therapeutic targets for treating leishmaniasis.
In order to use L. major promastigotes to study membrane repair and interactions with toxins, medium-throughput techniques are needed. While high-resolution live cell imaging enables the visualization of labeled proteins and membranes in real time, it is low throughput and may not measure cellular survival. Medium-throughput viability assays include dye uptake measured by flow cytometry, the measurement of mitochondrial activity, or the release of cellular proteins like lactate dehydrogenase (LDH). In mammalian cells, LDH assays do not quantitatively measure cell death20. Furthermore, population-based assays like LDH release or mitochondrial activity do not allow robust single-cell or multiparametric analysis20. In contrast, flow cytometry-based assays enable multiparametric single-cell analysis20. However, these assays have not been applied to understanding toxin biology or responses to toxins in L. major promastigotes.
In this study, SLO is used as a tool to understand the plasma membrane perturbation of the sphingolipid null mutant of L. major in two different buffers-the M199 media routinely used to culture L. major promastigotes and the simpler Tyrode&#39;s buffer. A medium-throughput flow cytometry assay is described and used to generate toxin dose-response curves. Data from the flow cytometric assay are modeled to a logistic curve to determine the LC50 values. With this information, a sublytic dose of SLO can be determined so that MAPK antibodies can be validated using western blotting.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.2 mL microtiter (Marsh) tubes</td>
			<td>Fisher</td>
			<td>02-681-376</td>
			<td>Cytotoxicity assay</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td>Toxin dilutions</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube&#160;</td>
			<td>Avantor VWR (Radnor, PA)</td>
			<td>89039-666</td>
			<td>To hold cells and media</td>
		</tr>
		<tr>
			<td>1x Phosphate buffered saline (PBS)</td>
			<td>Fisher</td>
			<td>BP399</td>
			<td>For cell processing</td>
		</tr>
		<tr>
			<td>3% H2O2&#160;</td>
			<td>Walmart&#160; (Fayetteville, AR)</td>
			<td>N/A</td>
			<td>For ECL</td>
		</tr>
		<tr>
			<td>5x M199</td>
			<td>Cell-gro</td>
			<td>11150067</td>
			<td>Basal growth media for L. major promastigotes</td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Baker</td>
			<td></td>
			<td>To culture cells in sterile conditions</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher</td>
			<td>BP1605-100</td>
			<td>Fraction V acceptable purity</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>BP510-100</td>
			<td>Stock concentration 100 mM</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher&#160;</td>
			<td>Heraeus Megafuge 40R</td>
			<td>To pellet the cells from culture</td>
		</tr>
		<tr>
			<td>Cy5 Mono-reactive dye pack</td>
			<td>Cytiva (Marlborough, MA)</td>
			<td>PA25031</td>
			<td>Fluorophore label for toxins</td>
		</tr>
		<tr>
			<td>Digital dry bath</td>
			<td>Benchmark</td>
			<td>BSH1002</td>
			<td>To denature protein samples</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Amresco</td>
			<td>0732-100G</td>
			<td>Stock concentration 0.5 M</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft (Redmond, VA)</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>Flow cytometer (4-laser Attune NxT)</td>
			<td>Fisher</td>
			<td></td>
			<td>Cytometer for data acquisition</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>BD (Ashland, OR)</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher</td>
			<td>BP531-500</td>
			<td>Fixative for counting cells</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Fisher</td>
			<td>BP673-1</td>
			<td>Selection agent for cells</td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Sigma</td>
			<td>Z805939</td>
			<td>Dilute 1:4 for cleaning cytometer</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher</td>
			<td>0267151B</td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>Human red blood cells</td>
			<td>Zen-bio (Durham, NC)</td>
			<td>SER-10MLRBC</td>
			<td>To validate toxin activity</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Nikon</td>
			<td>Eclipse 55i</td>
			<td>To visualize cells</td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Fisher</td>
			<td>88018</td>
			<td>For probing proteins via antibodies</td>
		</tr>
		<tr>
			<td>Pipettors and tips</td>
			<td>Avantor VWR</td>
			<td></td>
			<td>To dispense reagents</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>To run SDS-PAGE and transfers</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Biotium</td>
			<td>40016</td>
			<td>Stock concentration 2 mg/mL in water</td>
		</tr>
		<tr>
			<td>Protein ladder</td>
			<td>Bio-Rad</td>
			<td>161-0373</td>
			<td>To determine molecular weight of proteins</td>
		</tr>
		<tr>
			<td>SDS-PAGE Running Apparatus (Mini Protean III)</td>
			<td>Bio-Rad</td>
			<td>165-3302</td>
			<td>To separate proteins based on their size</td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>R&#38;D</td>
			<td>DY992</td>
			<td>To seal plates with cells</td>
		</tr>
		<tr>
			<td>Streptolysin O C530A plasmid insert</td>
			<td></td>
			<td></td>
			<td>Cloned into pBAD-gIII vector (Reference: 7)</td>
		</tr>
		<tr>
			<td>Streptolysin O C530A toxin</td>
			<td>Lab purified</td>
			<td></td>
			<td>Specific activity&#160; 4.34 x 105 HU/mg</td>
		</tr>
		<tr>
			<td>Swinging bucket rotor</td>
			<td>Thermo Fisher&#160;</td>
			<td>75003607</td>
			<td>To centrifuge cells</td>
		</tr>
		<tr>
			<td>V-bottom plate</td>
			<td>Greiner Bio-one</td>
			<td>651206</td>
			<td>For cytotoxicity assay</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Benchmark</td>
			<td>BV1000</td>
			<td>To mix cells</td>
		</tr>
		<tr>
			<td>Western blot imaging system (Chemi-doc)</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>To visualize proteins by western blot</td>
		</tr>
		<tr>
			<td>Western Blot Transfer Apparatus (Mini Protean III)</td>
			<td>Bio-Rad</td>
			<td>170-3930</td>
			<td>Transfer proteins to nitrocellulose</td>
		</tr>
		<tr>
			<td>Whatman Filter paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3030-700</td>
			<td>Used in transfer of proteins to nitrocellulose</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9102S</td>
			<td>Rabbit (1:1000 dilution)</td>
		</tr>
		<tr>
			<td>Anti-lipophosphoglycan (LPG) antibody</td>
			<td>CreativeBioLabs&#160;</td>
			<td>Cat# WIC79.3</td>
			<td>Mouse (1: 1000)</td>
		</tr>
		<tr>
			<td>Anti-MEK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9122L</td>
			<td>Rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP conjugate</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>Cat#715-035-151</td>
			<td>Donkey (1:10000)</td>
		</tr>
		<tr>
			<td>Anti-phosphoERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9101S</td>
			<td>Rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>Anti-pMEK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9121S</td>
			<td>Rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP conjugate</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>Cat#711-035-152</td>
			<td>Donkey (1:10000)</td>
		</tr>
		<tr>
			<td>Anti-tubulin antibody</td>
			<td>Sigma</td>
			<td>Cat# T5168</td>
			<td>Mouse (1: 2000)</td>
		</tr>
		<tr>
			<td>Leishmania major Genotypes&#160;</td>
			<td></td>
			<td></td>
			<td>Reference: 13</td>
		</tr>
		<tr>
			<td>Episomal addback (spt2-/+SPT2)</td>
			<td></td>
			<td></td>
			<td>&#916;spt2::HYG/&#916;spt2:PAC/+pXG-SPT2</td>
		</tr>
		<tr>
			<td>Serine palmitoyltransferase subunit 2 knockout (spt2-)</td>
			<td></td>
			<td></td>
			<td>&#916;spt2::HYG/&#916;spt2::PAC&#160;</td>
		</tr>
		<tr>
			<td>Wild type (WT)</td>
			<td></td>
			<td></td>
			<td>LV39 clone 5 (Rho/SU/59/P)</td>
		</tr>
	</tbody>
</table>

deciphering the molecular mechanism and function of pore-forming toxins using leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major,&#160;offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Increased promastigote sensitivity to SLO in Tyrode&#39;s buffer compared to M199 
The SLO sensitivity of L. major promastigotes was compared between different assay buffers. Wild-type, spt2-, and spt2-/+SPT2&#160;promastigotes were challenged with SLO in serum-free M199 or Tyrode&#39;s buffer supplemented with 2 mM CaCl2 for 30 min prior to analysis on a flow cytometer. Suitable parasites for analysis were single cells identified by forwar...

Watch this Scientific Journal Video about Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major at JoVE.com

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While ...

The mechanism of resistance to pore-forming toxins is poorly understood. Our protocol enables function and mechanism to pore-forming toxins using Leishmania major, a genetically tractable and physiologically relevant system. The main advantage of the cytotoxicity assay is comparing cell viability of multiple phenotypes at a single-cell level in a medium-throughput assay when challenged with toxins in real time.Membrane-perturbing agents like amphotericin B are frontline therapies for Leishmania. This assay enables the identification of agents that potentiate amphotericin B and/or new membrane-damaging agents. This assay provides insight into areas such as repair response and signaling pathways related to repair responses.This assay can be applied to protozoans such as Trypanosoma and Naegleria fowleri. People find it difficult to understand the toxin serial dilution series. So the best advice I can give is to draw a schematic of the serial dilution series before you set up the experiment.To begin, reserve 0.5 milliliters of processed promastigotes in a separate tube as unstained control. Add two milligrams per milliliter propidium iodide, or PI, to a final concentration of 10 micrograms per milliliter to the remaining promastigotes. Vortex for three seconds.Add processed promastigotes to each well of a V-bottom, 96-well plate or Marsh tubes, and place the plate or tube rack on the ice at an approximately 45-degrees angle from viewing. Add 100 microliters of assay buffer with propidium iodide to each no-toxin control. Verify that the control was correctly added by visually identifying tubes with a total volume of 200 microliters that appear darker in color.Add the volume of toxin to the highest dilution. Then, serially dilute the toxin. Pipette up and down at least eight times to ensure mixing.Starting from the lowest toxin concentration, quickly add 100 microliters of toxin to the correct row, and continue until all the toxin has been added to the cells. Seal the plate with sealing tape. After incubating at 37 degrees Celsius for 30 minutes, package and transport the plate to the flow cytometer.For data acquisition, start with setting up the flow cytometer and acquisition software according to the manufacturer's instructions and per facility policy. Using the unstained L.major promastigote sample, set the gates for forward and side scatter and the initial fluorescent parameters based on the dyes chosen. Using single-stained controls, set the gates for the viability dye and any fluorescently labeled toxins.Monitor forward scatter versus time for microclogs, and acquire more than 10, 000 gated events for each sample on the cytometer. For data analysis, gate total single-cell L.major promastigotes by gating on forward and side scatter and time as needed. Use height or area as recommended for the flow cytometer.Identify and gate dead cells as PI high. Organize the dose-response curve in Excel for analysis. Determine the average percent PI high for each condition between the two technical replicates.Include toxin concentration and average percent specific lysis, along with experimental details and/or raw percent PI high calculations. Label four more columns as Modeled, Residuals, Parameters, and Parameter Values. Verify that the first columns correspond to the experimental parameters, the toxin concentration, and percent specific lysis.Initialize the parameters L, k, and c by entering the values in the Parameter Values column. In the Modeled column, create the logistic model. In the Residuals column, calculate the square of the difference between the modeled number and the actual specific lysis.In the Parameter Values column, next to SUM, sum all the values in the Residuals column. In the Parameter Values column, next to LC50, initialize the equation to calculate the LC50 from the determined values. Open the Solver from the Data tab.Select the set objective to be the cell containing the sum of the residuals calculated. Set it to Min. Change the variable cells for the parameter values of L, k, and c.For negative k values, modify equations by factoring out negative one from k to change k to positive. Use the GRG Nonlinear Solving method, and click Solve. Check the curve and that the LC50 is automatically calculated.Verify the fit by graphically plotting both percent specific lysis and modeling against toxin concentration. The sphingolipid-deficient SPT2 knockout promastigotes were sensitive to SLO in both serum-free M199 and Tyrode's buffer. Wild type and SPT2 add-back promastigotes were resistant to SLO in serum-free M199 but had less than 20%specific lysis in Tyrode's buffer.The SPT2 knockout promastigotes were approximately eightfold more sensitive to SLO in Tyrode's buffer than in M199. These data demonstrate that toxin sensitivity may vary based on the buffer used. A 120-kilodalton band was observed for phospho-MEK in the L.major promastigotes.For total MEK, bands at about 120 kilodaltons and 55 kilodaltons were observed, which are consistent with the sizes of MRK1 and LmxMKK, respectively. Phospho-ERK detected similar bands, while the ERK antibody staining was not robust in this assay. The most important thing to remember when attempting this procedure is ensuring the proper handling of the toxin.Leishmania can be utilized as a genetically tractable model to study plasma membrane repair, providing new insights into membrane repair mechanics during Leishmania bacteria interaction in sandfly midgut.

deciphering-molecular-mechanism-function-pore-forming-toxins-using

Research

JoVE Journal

Immunology and Infection

1.3K visualizações.  Texas Tech University. O mecanismo de resistência às toxinas formadoras de poros é pouco compreendido. Nosso protocolo permite a função e o mecanismo para formar toxinas formadoras de poros usando Leishmania major, um sistema geneticamente tratável e fisiologicamente relevante. A principal vantagem do ensaio de citotoxicidade é comparar a viabilidade celular de múltiplos fenótipos em um nível de célula única em um ensaio de rendimento médio quando desafiado com toxinas em tempo real.Agentes pert...