a lactate dehydrogenase release assay to detect cryptococcus neoformans infection in macrophages

A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages

To prepare fungal cells for a macrophage infection, collect and centrifuge C. neoformans cells from a mid-log phase culture, followed by three gentle washes with 1 milliliter of room temperature PBS under the same centrifuge conditions.
After the last wash, re-suspend the fungal cells at a 1.2 times 10 to the eighth cells per milliliter, of antibiotic-free cell culture medium concentration, and add 1 milliliter of fungal cell suspension to each of 4 wells of a 70% to 80% confluent 6-well macrophage culture plate. Then, place the plate in the cell culture incubator for three hours. Carefully tilt the plate to allow each well to be gently washed three times with 1 milliliter of PBS per well per wash.
To measure the infection proficiency after the last wash, add 1 milliliter of antibiotic-free medium to each well of the 6-well plate, and place the plate in the cell culture incubator. At the appropriate experimental time points, collect the supernatant from each well, and measure the amount of LDH in each well according to standard protocols. At the same time points, lyse the uninfected macrophage cells to determine the value for maximum cytotoxicity.
The cytotoxicity can then be calculated using the formula as indicated.

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Immunology

Immune Response

Host Defense Mechanism

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1. Verification of infection
NOTE: Using a cytotoxicity assay, infection proficiency can be measured. The following protocol will highlight the application of a cytotoxicity product to measure LDH (lactate dehydrogenase) release. Other cytotoxicity products can also be used
<ol>
	<li>Preparation of C. neoformans infection of macrophage cells
	<ol>
		<li>Preparation of fungal cells
		<ol>
			<li>Using a glycerol stock, streak wildtype C. neoformans strain (H99) onto Yeast-extract peptone dextrose (YPD) agar plate to isolate single colonies.</li>
			<li>Incubate overnight for 16 h at 37 &#176;C in a static incubator.</li>
			<li>Select a single colony of wildtype C. neoformans strain and culture in 5 mL of YPD broth in a loosely capped 10 mL test tube. Perform in quadruplicate.</li>
			<li>Incubate overnight for 16 h at 37 &#176;C in a shaking incubator at 200 rpm.</li>
			<li>The following day subcultures each overnight culture in a 1:100 dilution into 3 mL YPD broth.</li>
			<li>Measure Optical density (OD600nm) values of fungal culture to determine the mid-log phase. Depending on the spectrophotometer, C. neoformans wild-type strain reaches the mid-log phase commonly following 6 to 8 h incubation in YPD with general OD600nm values ranging from 1.0 to 1.5.</li>
			<li>Once cells reach the mid-log phase, take a 10 &#181;L aliquot of fungal cell suspension into a clean 1.5 mL microcentrifuge tube and dilute 1:100 in sterile 1x phosphate-buffered saline (PBS). Count the number of cells using a hemocytometer.</li>
			<li>Collect and centrifuge cells at 1,500 x g for 10 min, gently wash the pellet with sterile room-temperature PBS, and repeat for a total of three washes.</li>
			<li>Resuspend cells in an antibiotic-free cell culture medium to achieve a concentration of 1.2 x 108 cells/mL.</li>
		</ol>
		</li>
		<li>Preparation of macrophage cells
		<ol>
			<li>Visualize each well in the 6-well plate to ensure that cells have reached 70-80% confluence. Alternatively, cells can be measured to achieve approx. 1.2 x 106 macrophage cells per well.</li>
			<li>Warm antibiotic-supplemented medium to 37 &#176;C prior to cell culture work. Ensure the work environment is sterilized prior to cell culture work. Visualize cells using a light microscope to ensure cells are adherent and healthy.</li>
			<li>Remove the cell culture medium from the dish either with a serological pipette or a vacuum aspirator.</li>
			<li>Gently add 5-7 mL of sterile room-temperature PBS (phosphate-buffered saline) to the 60 x 15 mm dish.</li>
			<li>Gently tilt the dish to wash adhered cells.</li>
		</ol>
		</li>
		<li>Co-culture of C. neoformans and macrophage cells
		<ol>
			<li>Add 1 mL of the resuspended C. neoformans cells to 4 wells containing macrophage cells. 
			NOTE: The number of plates required will need to be calculated prior to beginning the experiment. Add 1 mL of antibiotic-free medium to empty wells.</li>
			<li>Allow cells to incubate at 37 &#176;C with 5% CO2 for 3 h.</li>
			<li>Remove the cell culture medium from the plate either with a serological pipette or a vacuum aspirator.</li>
			<li>Gently add 1 mL of sterile room-temperature PBS.</li>
			<li>Gently tilt the plate to wash non-attached or non-phagocytosed extracellular C. neoformans cells.</li>
			<li>Add 1 mL of antibiotic-free medium to each well in the 6-well plate.</li>
			<li>Incubate cells at 37 &#176;C with 5% CO2.</li>
		</ol>
		</li>
		<li>At selected time points (e.g., 1, 3, 6, 12, and 24 hours) collect supernatant for measurement of LDH release according to the manufacturer&#39;s instructions.</li>
		<li>At the same time points, uninfected macrophage cells will be lysed to a determined value for maximum cytotoxicity.</li>
		<li>Calculate cytotoxicity as follows: 
		<img alt="Equation 1" src="/files/ftp_upload/21660/21660_Equation1.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mM Tris-HCl, pH 8.5</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Maintain at 4&#176;C</td>
		</tr>
		<tr>
			<td>60 x 15 mm Dish, Nunclon Delta</td>
			<td>ThermoFisher Scientific</td>
			<td>174888</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>ThermoFisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical falcon tubes (15 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>ThermoFisher Scientific</td>
			<td>AMQAX1000</td>
			<td>Not essential, haemocytometer can be used as an alternative.</td>
		</tr>
		<tr>
			<td>CytoTox 96 Non-Radioactive Cytotoxicity Assay</td>
			<td>Promega</td>
			<td>G1780</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>12483020</td>
			<td>Heat inactivate by incubating at 60&#176;C for 30 minutes. Prepare 50 ml aliquots and flash freeze. Thaw prior to media preparation</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20&#176;C until use. Discard after each use (do not freeze-thaw repeatedly).</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>25030081</td>
			<td>Can be aliquot and frozen for storage. Thaw prior to media preparation.</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>13864457</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin : Streptomycin 10k/10k</td>
			<td>VWR</td>
			<td>CA12001-692</td>
			<td>Can be aliquot and frozen for storage. Thaw prior to media preparation.</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>VWR</td>
			<td>CA12001-676</td>
			<td>Purchase not required. PBS can also be prepared but sterile filteration must be performed before use.</td>
		</tr>
		<tr>
			<td>Pipette, Disposable Serological (10 mL)</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, Disposable Serological (25 mL) Basix</td>
			<td>Fisher Scientific</td>
			<td>14955235</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C protease mix, MS grade</td>
			<td>Pierce</td>
			<td>A40007</td>
			<td>Maintain at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>U1250-1KG</td>
			<td>Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20 &#176;C until use. Discard after each use (do not freeze-thaw repeatedly).</td>
		</tr>
		<tr>
			<td>Yeast-extract peptone dextrose broth</td>
			<td>BD Difco</td>
			<td>BM20</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Ball, B., et al. <a href="https://app.jove.com/v/61881">Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions.</a> J. Vis. Exp. (2020)
In this video, we demonstrate a cytotoxicity assay to assess Cryptococcus neoformans, a pathogenic fungus, infection in macrophages. The cytotoxicity is determined by the release of cytoplasmic lactate dehydrogenase enzyme due to macrophage lysis caused by Cryptococcus neoformans infection.

1. Verification of infection
NOTE: Using a cytotoxicity assay, infection proficiency can be measured. The following protocol will highlight the ...

Take a multi-well plate containing macrophages.
Add Cryptococcus neoformans to the test wells, while the control wells contain only macrophages.
Macrophages phagocytose fungi within phagosomes.
Wash with buffer to remove non-phagocytosed fungi. Add antibiotic-free media. Incubate.
The phagosome merges with the lysosome, forming a phagolysosome. Fungi modulate phagolysosome pH and possess intrinsic mechanisms to survive harsh phagolysosomal conditions. Fungi permeabilize the phagolysosomal membrane and replicate, accompanied by cytosolic accumulation of polysaccharide-filled vesicles.
Intracellular fungal residence and replication cause cellular damage, triggering cell-death pathways. This causes macrophage lysis and intracellular content release, including lactate dehydrogenase or LDH, into media.
Collect LDH-containing media at different time points. Lyse the control macrophages at similar time points for maximum cellular LDH release. Add the assay mixture to the collected media and lysates. LDH converts lactate to pyruvate, reducing NAD+. Diaphorase reduces tetrazolium salt, forming a red formazan product.
Using a plate reader, measure the formazan absorbance to quantify macrophage lysis following Cryptococcus neoformans infection.

45 vues. Un test de libération de lactate déshydrogénase pour détecter l’infection à Cryptococcus neoformans dans les macrophages

Vidéo: Un test de libération de lactate déshydrogénase pour détecter l’infection à Cryptococcus neoformans dans les macrophages - Expérience

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Size Matters : Mesure du diamètre de la Capsule à Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

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A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages

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