eoe sub articles

EoE Sub Articles

1. K562 Killing Assay
NOTE: K562 cells should be grown in culture at 37 &#176;C and 5% CO2 with RPMI complete medium until they reach 80% confluence before the assay.
<ol>
	<li>Prepare a 5 mM carboxyfluorescein succinimidyl ester (CFSE) stock solution by adding 18 &#181;l of DMSO to the vial.</li>
	<li>Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well to achieve the desired equi-nicotine units or nicotine concentrations for each well. 
	CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs into RPMI complete medium at a concentration of 1.5 x 106 cells/well. The total volume of cells with WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>Cover the plate and incubate for 3 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the K562 cells by adding 10 ml of DPBS and centrifuge at 400 x g for 8 min at RT.</li>
	<li>Resuspend the cells with 10 ml of DPBS and count the K562 cells.</li>
	<li>Prepare a CFSE working solution by adding 1 &#181;l of CFSE stock solution to 1 ml DPBS.</li>
	<li>Add 1 ml of the CFSE working solution to 1 ml of the K562 cell suspension containing 1 - 2 x 107 cells. Vortex and incubate precisely for 2 min at RT.</li>
	<li>Immediately add 200 &#956;l of FBS. Vortex and incubate precisely for 2 min at RT.</li>
	<li>Add 10 ml of RPMI complete medium and centrifuge the tube at 400 x g for 8 min at RT.</li>
	<li>Remove the RPMI supernatant, and break the pellet, and resuspend the cells with 10 ml of RPMI complete medium, and count the CFSE-labeled K562 cells.</li>
	<li>Wash the PBMCs by centrifuging the plate at 300 x g for 3 min at RT. Aspirate the supernatant by decanting the liquid. Replace the cover and vortex the bottom of the plate. Add 200 &#181;l of RPMI complete medium and repeat the washing step one more time.</li>
	<li>Add CFSE-labeled K562 cells at a ratio of 1:15 (100,000 K562s:1.5 x 106 PBMCs) to each sample well of the PBMC plate and further incubate for 5 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the cell mix by centrifuging at 300 x g for 3 min at RT. Aspirate the supernatant by discarding the liquid. Place the cover and vortex the bottom of the plate.</li>
	<li>Add 200 &#181;l of running buffer and repeat the washing step described in step 14 one more time.</li>
	<li>Add 95 &#181;l of running buffer followed by 5 &#181;l of 7AAD to each well for a total volume of 100 &#181;l/well. Incubate the plate in the dark at RT for 15 min.</li>
	<li>Add 100 &#181;l of running buffer to each well and transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on the flow cytometer.</li>
	<li>Determine the percentage of 7AAD-positive and CFSE-positive cells using flow cytometry analysis software.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 X 75 tubes</td>
			<td>BD Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>500 ml bottle</td>
			<td>Corning</td>
			<td>430282</td>
			<td></td>
		</tr>
		<tr>
			<td>7AAD</td>
			<td>BD Pharmingen</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well flat bottom plate</td>
			<td>Termo Nunc</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round bottom plates</td>
			<td>BD Falcon</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>58110R</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Molecular Probes Life Technologies</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Cluster tubes</td>
			<td>Corning</td>
			<td>4401</td>
			<td>Harmful if swallowed, carcinogen</td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl sulfoxide )</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter unit</td>
			<td>Nalgene</td>
			<td>156-4020</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>FACS Canto II</td>
			<td>8 colors at Ex 405 nm and Em785 nm.</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>FACS Calibur</td>
			<td>4 colors at Ex 495 nm and Em 785 nm.</td>
		</tr>
		<tr>
			<td>Flow cytometry analysis software</td>
			<td>Tree Star</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotine</td>
			<td>Sigma-Aldrich</td>
			<td>N3876</td>
			<td>Acute toxicity, Environmental hazard</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>&#34;M&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Flammable, Skin irritation</td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Gibco Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Running buffer</td>
			<td>MACS Running Buffer, Miltenyi Biotech</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
	</tbody>
</table>

killing k562 cells by peripheral blood mononuclear cells exposed to tobacco product preparations

Source: Arimilli, S. et al., <a href="https://app.jove.com/v/52351">Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations.</a> J. Vis. Exp. (2015)
In this video, we study the effect of a whole smoke-conditioned medium, a combustible tobacco product preparation, on K562 cell killing by effector peripheral blood mononuclear cells.

Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations

Source: Arimilli, S. et al., Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations. J. Vis. Exp. (2015)
In ...

Tumor cells activate effector immune cells to release cytokines and cytolytic granules containing perforin and granzymes. Perforin forms pores in the tumor cell membrane, facilitating granzyme entry into the cell and initiating apoptosis, causing cell death.
To analyze the cytolytic function of human peripheral blood mononuclear cells, PBMCs, following tobacco product preparation, TPP, exposure, take a multi-well plate with increasing TPP concentrations. A medium-containing well acts as the control.
Add PBMC suspension. TPPs&#39; primary immunosuppressive agent, nicotine, binds to effector PBMCs&#39; nicotinic acetylcholine receptors, downregulating the pro-inflammatory cytokine production.
Centrifuge. Remove the TPP-containing supernatant. Resuspend the PBMCs in media. Add fluorescently labeled K562 cells, human leukemic cells having an increased susceptibility to PBMC effector cell cytotoxicity, to the wells.&#160;
TPP-treated effector cells with suppressed immune responses exhibit reduced perforin expression and cytokine release, decreasing the PBMC&#39;s cytolytic ability.
Centrifuge. Resuspend the cells in a buffer followed by a cell-impermeable fluorescent dye. The dye enters the cells with compromised membranes and stains the DNA.
Using a flow cytometer, determine the proportion of dead and live K562 cells in the wells. A lower percentage of dead K562 cells in wells with higher TPP concentrations than the control indicates suppression of K562 cell killing by TPP-treated PBMCs.

killing-k562-cells-peripheral-blood-mononuclear-cells-exposed-to

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Immunology

Immune Response

Immune Modulation

142 Views. Source: Arimilli, S. et al., Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations. J. Vis. Exp. (2015) In this video, we study the effect of a whole smoke-conditioned medium, a combustible tobacco product preparation, on K562 cell killing by effector peripheral blood mononuclear cells. 

Video: Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations - Concept

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

JoVE Journal

Immunology and Infection

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations

Transcript

Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)