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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Obtaining invariant natural killer T cells (iNKT Cells) with Adoptive Cellular Therapy
<ol>
	<li>Directional induction of iNKT cells
	<ol>
		<li>Inject normal mice intraperitoneally with &#945;-Galactosylceramide (&#945;-GalCer) (0.1 mg/kg of body weight).</li>
	</ol>
	</li>
	<li>Isolation of iNKT cells
	<ol>
		<li>Three days after modeling, inject mice intraperitoneally with 1% sodium pentobarbital (50 mg/kg of body weight) for anesthetization. Adequately anesthetized mice do not show a hind paw withdrawal response to a toe pinch.</li>
		<li>Isolate the spleen of a DBA/1 mouse after injecting it intraperitoneally with &#945;-GalCer. Prepare a single-cell suspension by cutting and grinding the spleen in a 200-mesh sieve.</li>
		<li>Wash the cell suspension with phosphate-buffered saline (PBS), centrifuge at 200 x&#160;g&#160;for 5 min, and discard the supernatant. Repeat.</li>
		<li>Resuspend the cells with 1 mL of whole blood and tissue dilution solution. Add 3 mL of mouse lymphocyte separation medium, then centrifuge the cells for 20 min at 300 x&#160;g&#160;at room temperature.</li>
		<li>Collect the layer of milky white lymphocytes (i.e., the second layer from the top), wash it 2x with PBS, and count with an automated cell counter.</li>
	</ol>
	</li>
	<li>Magnetic activated cell sorting (MACS) positive&#8194;selection&#8194;strategy for purification of iNKT cells 
	NOTE: For the pretreatment of CD1d tetramers, 1 mg/mL of &#945;-Galcer was diluted to 200 &#956;g/mL with 0.5% of Tween-20 and 0.9% of NaCl, and 5 &#181;L of the resulting solution was added to 100 &#181;L of the CD1d tetramer solution. The mixture was incubated for 12 h at room temperature and placed at 4 &#176;C for use. T cell receptor (TCR) &#946; was diluted 80x with deionized water. All other antibodies were used as a stock solution.
	<ol>
		<li>Resuspend 107&#160;cells with 100 &#181;L of 4 &#176;C PBS, add 10 &#181;L of &#945;-GalCer-loaded CD1d Tetramer-phycoerythrin&#160;(CD1d Tetramer-PE), and incubate them at 4 &#176;C for 15 min in the dark.</li>
		<li>Wash the cells 2x with PBS and resuspend them in 80 &#181;L of PBS.</li>
		<li>Add 20 &#181;L of anti-PE-MicroBeads and incubate them at 4 &#176;C for 20 min in the dark.</li>
		<li>Wash them 2x with PBS and resuspend the cells with 500 &#181;L of PBS.</li>
		<li>Place the sorting column in the magnetic field of the MACS sorter and rinse with 500 &#181;L of PBS.</li>
		<li>Add the cell suspension from step 1.3.4 to the sorting column, collect the flowthrough, and rinse 3x with PBS buffer.</li>
		<li>Remove the magnetic field and collect the cells from the sorting column. At this point, add 1 mL of PBS buffer to the sorting column and quickly push the plunger at a constant pressure to drive the labeled cells to the collection tube and obtain purified iNKT cells. Count with an automated cell counter.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-PE MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-048-801</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Columns</td>
			<td>Miltenyi</td>
			<td>MS</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Cryogenic Centrifuge</td>
			<td>Beckman</td>
			<td>Allegra&#174; X-15R</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Mouse CD1d Tetramer-PE</td>
			<td>MBL</td>
			<td>TS-MCD-1</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>Mouse percoll</td>
			<td>Solarbio</td>
			<td>P8620</td>
			<td>China</td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Olympus</td>
			<td>Olympus-II</td>
			<td>Japan</td>
		</tr>
	</tbody>
</table>

isolating invariant natural killer t cells from a mouse model

Source: Meng, M., et al. <a href="https://app.jove.com/v/60048">Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice.</a>&#160;J. Vis. Exp.&#160;(2020)
This video illustrates a method for isolating invariant natural killer T cells, also known as iNKT cells. The procedure begins with injecting a synthetic glycolipid into a mouse to stimulate the activation and proliferation of iNKT cells in the spleen. Subsequently, single-cell suspensions are prepared from the spleen and incubated with iNKT-cell-recognizing complexes labeled with fluorophores, followed by magnetic bead-based separation, to isolate the iNKT cells.

Isolating Invariant Natural Killer T Cells from a Mouse Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin by intraperitoneally injecting a synthetic glycolipid into a mouse.
Antigen-presenting cells, or APCs, internalize the glycolipid and present it via the non-classical MHC molecule CD1d.
The APCs migrate to the spleen and interact with the invariant natural killer T cells or iNKT cells, inducing iNKT cell activation and proliferation.
Harvest the spleen. Mechanically dissociate it to generate a single-cell suspension.
Transfer the cells to a tube. Add a density gradient medium and centrifuge to separate the lymphocytes.
Collect the lymphocytes. Add glycolipid antigen-loaded fluorophore-labeled CD1d tetramers that interact with the iNKT cells.
Remove unbound tetramers. Overlay with magnetic microbeads that bind to the tetramer-linked fluorophores.
Remove unbound beads and load the cell suspension onto a magnetic column.
Under the magnetic field, the bead-bound iNKT cells are retained in the column while the remaining cells flow through.
Remove the column from the magnetic field. Add an elution buffer to elute the iNKT cells.

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141 次观看. 来源:Meng， M. 等人 葡萄糖-6-磷酸异构酶 （G6PI） 诱导的 RA 小鼠中 iNKT 细胞的过继免疫疗法。J. Vis. Exp.（2020 年） 该视频演示了一种分离不变的自然杀伤 T 细胞（也称为 iNKT 细胞）的方法。该程序首先将合成糖脂注射到小鼠体内，以刺激脾脏中 iNKT 细胞的活化和增殖。随后，从脾脏制备单细胞悬液，并与用荧光团标记的 iNKT 细胞识别复合物一起孵育，然后进行基于磁珠的分离?...

视频: 从小鼠模型中分离不变的自然杀伤 T 细胞 - 概念

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

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JoVE Journal

免疫与感染

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

整合影像流式细胞仪和转录组分析, 评估改变的内吞 CD1d 贩运

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Isolating Invariant Natural Killer T Cells from a Mouse Model

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Isolating Invariant Natural Killer T Cells from a Mouse Model