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EoE Sub Articles

1. Cytotoxicity Assay: Assay Protocol
<ol>
	<li>Use a round bottom, culture treated 96-well plate to set up the plate as suggested in Table 1. This table shows the experimental controls and 3 sets of NK experimental columns. This can be expanded to a total of 10 NK experimental columns in a 96-well plate.</li>
	<li>Centrifuge the assay plate at 250 x g for 4 min to be certain that the effector and target cells are in contact. Incubate the 96-well plate for 5 hours in a humidified chamber incubator at 37 &#176;C, 5% CO2 to achieve ample contact between target and effector cells and target cell lysis by effector cells. 
	NOTE: The protocol can be paused here.</li>
	<li>45 min prior to harvesting supernatants, add 10 &#956;L of 10x Lysis Solution to the Target Cell Maximum LDH Release wells (Wells 1E, 1F, 1G, and 1H) and place the plate back in the humidified chamber.</li>
	<li>After the incubation time is completed, centrifuge the plate at 250 x g for 4 min. Using a multichannel pipettor, transfer 50 &#956;L aliquots from all wells to a fresh 96-well flat-bottom assay plate. 
	NOTE: Do not touch the bottom of the wells so that cells are not transferred into the fresh assay plate. Do not use a cultured treated plate as the fresh assay plate.</li>
</ol>
2. Make Assay Reagent.
<ol>
	<li>After Assay Buffer has reached room temperature, add 12 mL of Assay Buffer to one Substrate Mix bottle. Invert and shake gently until completely dissolved. 1 bottle is enough for two 96-well plates. 
	NOTE: Assay Buffer should be protected from light while being thawed. Mix immediately prior to use.</li>
	<li>Add 50 &#956;L of Assay Reagent to all wells in the assay plate from step 4.6. Cover the plate with foil to protect it from light and incubate for 30 min at room temperature. 
	NOTE: Newly made Assay Reagent should be stored in a freezer. Reagent is good for approximately 8 weeks.</li>
	<li>Add 50 &#956;L of Stop Solution to each well. Read plate within 1 hour after adding Stop Solution and record the absorbance at 490 nm. 
	Representative data is shown in Table 2.</li>
</ol>
Table 1: Assay Plate Layout. TCS = Target cell Spontaneous LDH Release (50 &#956;L target cells + 50 &#956;L target cell medium); TCM = Target Cell Maximum LDH Release (50 &#956;L target cells + 50&#956;L target cell medium); VCC = Volume Correction Control (50 &#956;L target cell medium + 50 &#956;L NK Cell medium); CMB = Culture Medium Background Control (50 &#956;L target cell medium + 50 &#956;L NK Cell medium); ECSR = Effector Cell Spontaneous Release (50 &#956;L NK cells + 50 &#956;L NK cells medium); EW = Experimental wells (50 &#956;L target cells + 50 &#956;L NK cells).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
		</tr>
		<tr>
			<td>TCS</td>
			<td>VCC</td>
			<td>ECSR-1</td>
			<td>ECSR-2</td>
			<td>ECSR-3</td>
		</tr>
		<tr>
			<td>TCS</td>
			<td>VCC</td>
			<td>ECSR-1</td>
			<td>ECSR-2</td>
			<td>ECSR-3</td>
		</tr>
		<tr>
			<td>TCS</td>
			<td>VCC</td>
			<td>ECSR-1</td>
			<td>ECSR-2</td>
			<td>ECSR-3</td>
		</tr>
		<tr>
			<td>TCS</td>
			<td>VCC</td>
			<td>ECSR-1</td>
			<td>ECSR-2</td>
			<td>ECSR-3</td>
		</tr>
		<tr>
			<td>TCM</td>
			<td>CMB</td>
			<td>EW-1</td>
			<td>EW-2</td>
			<td>EW-3</td>
		</tr>
		<tr>
			<td>TCM</td>
			<td>CMB</td>
			<td>EW-1</td>
			<td>EW-2</td>
			<td>EW-3</td>
		</tr>
		<tr>
			<td>TCM</td>
			<td>CMB</td>
			<td>EW-1</td>
			<td>EW-2</td>
			<td>EW-3</td>
		</tr>
		<tr>
			<td>TCM</td>
			<td>CMB</td>
			<td>EW-1</td>
			<td>EW-2</td>
			<td>EW-3</td>
		</tr>
	</tbody>
</table>
Table 2: Raw absorbance data of an assay plate containing the experimental controls in columns 1 and 2 and experimental wells to measure the cytotoxicity of placental NK cells from NP and RUPP rats against YAC1 target cells.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>NP</td>
			<td>RUPP</td>
			<td>NP</td>
		</tr>
		<tr>
			<td>0.615</td>
			<td>0.484</td>
			<td>0.473</td>
			<td>&#160;0.463</td>
			<td>0.471</td>
		</tr>
		<tr>
			<td>0.586</td>
			<td>0.484</td>
			<td>0.458</td>
			<td>0.474</td>
			<td>&#160;0.469</td>
		</tr>
		<tr>
			<td>0.61</td>
			<td>0.486</td>
			<td>0.464</td>
			<td>0.459</td>
			<td>0.469</td>
		</tr>
		<tr>
			<td>0.605</td>
			<td>0.55</td>
			<td>0.524</td>
			<td>0.526</td>
			<td>0.543</td>
		</tr>
		<tr>
			<td>0.576</td>
			<td>0.504</td>
			<td>0.519</td>
			<td>0.502 0.528</td>
			<td>00.528</td>
		</tr>
		<tr>
			<td>0.565</td>
			<td>0.512</td>
			<td>0.515</td>
			<td>0.513</td>
			<td>0.531</td>
		</tr>
		<tr>
			<td>0.563</td>
			<td>0.581</td>
			<td>0.526</td>
			<td>0.508</td>
			<td>0.519</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well Tissue Culture Plate</td>
			<td>CELLTREAT</td>
			<td>229190</td>
			<td>Sterile, Round Bottom</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher</td>
			<td>8100241</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotox 96 Non-Radioactive Cytotoxicity Assay Kit</td>
			<td>Promega</td>
			<td>G1780</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>11875135</td>
			<td>1640 Medium</td>
		</tr>
		<tr>
			<td>YAC-1 cell</td>
			<td>ATCC</td>
			<td>TIB-160</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro assay for evaluating natural killer cell cytotoxicity against cancer cells

Source: Baik, C. H., et al. <a href="https://app.jove.com/v/58961">A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function.</a> J. Vis. Exp. (2019)
This video demonstrates performing a lactate dehydrogenase release assay to determine the cytotoxicity of natural killer (NK) cells against the target cancer cells. The assay quantifies the release of the cytoplasmic lactate dehydrogenase enzyme from cancer cells by measuring the absorbance of the red-colored product formed.

An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

1. Cytotoxicity Assay: Assay Protocol

	Use a round bottom, culture treated 96-well plate to set up the plate as suggested in Table 1. This table shows ...

Take a multi-well plate containing natural killer, or NK, cells and cancer cells. Control wells contain only cancer cells.
Centrifuge to facilitate cellular contact.
Incubate. Activating receptors on NK cells bind to cancer cell surface ligands, triggering NK cells to release granules containing cytotoxic molecules.
These molecules create cancer cell membrane pores and induce apoptosis releasing intracellular contents, including lactate dehydrogenase, or LDH.
Add a detergent to lyse the cancer cells in control wells for maximum LDH release.
Centrifuge and transfer a portion of LDH-containing supernatants to an assay plate.
Add the assay reagent and incubate.
LDH converts lactate to pyruvate reducing NAD+. Diaphorase uses NADH&#160; to reduce tetrazolium salt, forming a red-colored formazan product.
Add an acidic solution to stop the enzymatic reaction.
Use a microplate reader to measure the formazan absorbance in the wells.
The formazan intensity is proportional to the number of lysed cancer cells in the test well,&#160; indicating NK cell cytotoxicity against cancer cells.

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Immunology

Immunotherapy

Cellular Immunotherapy

159 Views. Source: Baik, C. H., et al. A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function. J. Vis. Exp. (2019) This video demonstrates performing a lactate dehydrogenase release assay to determine the cytotoxicity of natural killer (NK) cells against the target cancer cells. The assay quantifies the release of the cytoplasmic lactate dehydrogenase enzyme from cancer cells by measuring the absorbance of the red-colored product formed. 

Video: An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells - Concept

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

JoVE Journal

Biochemistry

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Immunology and Infection

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

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