an in vitro technique for uv irradiation-mediated generation of apoptotic t cells

An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells

Begin by culturing Jurkat T cells in basal cell culture medium, supplemented according to the manuscript directions, at 37 degrees Celsius and 5% carbon dioxide. Jurkat T cells are a suspension cell line that can be maintained through passaging into pre-warmed culturing media every three days.
Prepare apoptotic cells by growing them to 90% confluency, which takes three to four days after passaging. Grow cells in five T75 flasks, to obtain a sufficient number of cells for this protocol. Once the cells are ready, aspirate the flask contents about 24 milliliters, and transfer them to a 50-milliliter conical tube. Count the cells using trypan blue staining and a hemocytometer.
Pellet the cells by centrifugation at 271 times g for five minutes, and discard the supernatant. Then, resuspend the cells in media to obtain three million cells per milliliter. Aliquot the cells into 100-by-20 millimeter tissue culture dishes by transferring five milliliters of cells per dish.
Prepare approximately nine culture dishes. Set one dish aside as an unexposed control, and expose the remaining dishes to UV. Set the UV crosslinker to the correct energy level by pressing the energy button and entering &#34;600&#34; with the number pad. Irradiate all dishes with the cells except for the control, making sure to remove the top cover of the tissue culture dish during UV exposure.
Then, incubate all the dishes in a cell culture incubator at 37 degrees Celsius and 5% carbon dioxide for four hours. After the incubation, combine all the irradiated cells into a 50-milliliter conical tube, and pellet them by centrifugation at 271 times g for five minutes. Discard the supernatant, and resuspend the cells in 24 milliliters of sterile PBS. Centrifuge the tube again, and remove the supernatant.

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1. Preparation of Jurkat T cell line (Day 2)
NOTE: All procedures should be conducted in a class II biological safety cabinet.
<ol>
	<li>Culture Jurkat T cells in 24 mL of basal cell culture medium + 10% FBS + 5% penicillin/streptomycin at 37 &#176;C + 5% CO2 (Table of Material). Jurkat T cells are a suspension cell line that can be maintained through passaging 1:6-1:8 into pre-warmed culturing media every 3 days. Do not shake.</li>
	<li>To prepare apoptotic cells, grow them to 90% confluency in each flask (which takes 3-4 days to achieve after passaging). For this study, use cells from five T75 flasks to obtain a sufficient number of cells used in this protocol. 
	NOTE: A confluent flask contains about 20-24 million cells.</li>
	<li>Pipette up cells (which is the entire flask) from each flask (approximately 24 mL) and transfer cells to a sterile 50 mL conical tube using a serological pipette. Use multiple conical tubes for multiple flasks.</li>
	<li>Count cells by removing an 11 &#181;L aliquot of cells from the 50 mL conical tube and mix with 11 &#181;L of trypan blue stain and pipette 11 &#181;L onto hemocytometer slides.</li>
	<li>Insert the slide into an automated cell counter and record the number of live cells to calculate the total cell count in each flask by multiplying the number of live cells by 24, as each flask contains 24 mL of media.</li>
	<li>Centrifuge the cell suspension at 271 x g for 5 min at room temperature (RT) to pellet cells.</li>
	<li>Discard the supernatant by aspiration and resuspend the cell pellet in media to obtain 3.0 x 106 cells per mL.</li>
	<li>Aliquot 5 mL of cells in 100 mm x 20 mm tissue culture dishes (approximately nine dishes will be used; the total amount of cells in each dish should be ~15 x 106).</li>
	<li>Use one dish for control/unexposed, and the remaining dishes will be exposed to UV.</li>
	<li>Set the UV crosslinker to the correct energy level, press the energy button, and enter &#34;600&#34; using the number pad, which the machine will read as 600 &#181;J/cm2 x 100. 
	NOTE: UV crosslinker energy units are in &#181;J/cm2 x 100; therefore, to achieve 60 millijoules/cm2, convert units to match the UV crosslinker.</li>
	<li>Irradiate all dishes with cells, not including the control, at 60 millijoules (mJ)/cm2 using the UV crosslinker. Remove the top cover of the tissue culture dishes during UV exposure, as UV light will not penetrate the plastic cover.</li>
	<li>Incubate all the dishes in a cell culture incubator, including unexposed control, at 37 &#176;C at 5% CO2 for 4 h.</li>
	<li>Confirm apoptosis by flow cytometry using an apoptosis assay detection kit containing annexin V and propidium iodide (PI) (markers for apoptosis and necrosis, respectively) after 4 h of incubation, per the manufacturer&#39;s instructions. 
	NOTE: Irradiating Jurkat T cells in the UV crosslinker at an energy level of 600 &#181;J/cm2, following a 4 h incubation will lead to &#8805;75% of apoptotic (both early and late) cells having more early apoptotic phenotype than the late apoptotic phenotype. This makes it easier for alveolar macrophages to recognize them and engulf them as their membranes are uncompromised, unlike late apoptotic cells, leading to a higher efferocytic index and more accurate imaging of alveolar macrophage efferocytosis in this study.
	<ol>
		<li>Pool 333 &#181;L (1 x 106 cells) of Jurkat T cells from several dishes (both &#34;no UV&#34; and &#34;UV-exposed&#34;) together to use for compensation analysis tubes.</li>
		<li>Aliquot 333 &#181;L of Jurkat T cells in an unstained, annexin V single stain, PI single stain, no UV control, and 600 &#181;J/cm2 UV-exposed labeled flow cytometry tubes.</li>
		<li>Centrifuge tubes at 188 x g for 5 min at RT and decant the supernatant.</li>
		<li>Wash cells by resuspending in 500 &#181;L of cold, 1x phosphate-buffered saline (PBS).</li>
		<li>Centrifuge and pellet cells at 188 x g for 5 min at RT. Discard the supernatant after centrifugation.</li>
		<li>Prepare 400 &#181;L of 1x binding buffer per flow tube by diluting 10x binding buffer with distilled water while cells are centrifuging.</li>
		<li>Prepare annexin V and PI incubation reagent (100 &#181;L per sample/tube) per the manufacturer&#39;s instructions.</li>
		<li>Decant the supernatant after centrifugation and gently resuspend all tubes in 400 &#181;L of 1x binding buffer, then add 100 &#181;L of annexin V incubation reagent to each sample tube. Lastly, add 100 &#181;L of annexin V single stain and PI single stain to their respective tubes, but do not add anything beyond the 1x binding buffer to the unstained tube.</li>
		<li>Incubate tubes in the dark for 15 min at RT.</li>
		<li>Centrifuge all cells at 188 x g for 5 min at RT and decant supernatant.</li>
		<li>Resuspend cells in 400 &#181;L of 1x binding buffer, then analyze samples for apoptosis by flow cytometry. Collect at least 10,000 events per tube to allow accurate representation of staining.</li>
	</ol>
	</li>
	<li>Combine all the irradiated cells from dishes into a 50 mL conical tube and pellet cells by centrifugation at 271 x g for 5 min at RT.</li>
	<li>Discard the supernatant from the tube by aspiration and resuspend cells in 24 mL of sterile phosphate-buffered saline (PBS) and pellet cells by centrifugation at 271 x g for 5 min at room temperature.</li>
	<li>Discard the supernatant from the tube by aspiration and resuspend cells in the amount of PBS used for dosing mice approved by IACUC. The dose used is between 5-10 x 106 cells/50 &#181;L per mouse; therefore, for 10 mice, resuspend in 500 &#181;L (the number of cells in each dose varies depending on how many cells are cultured for irradiation). 
	NOTE: Make at least two additional doses to account for any liquid that may stick to the sides of the pipette tip resulting in the loss of cells.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Annexin V-FITC Kit</td>
			<td>Trevigen</td>
			<td>4830-250-K</td>
			<td>The TACS Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells when using flow cytometry.</td>
		</tr>
		<tr>
			<td>BCL2 Jurkat T Cells</td>
			<td>ATCC</td>
			<td>ATCC CRL-2899</td>
			<td>The BCL2 Jurkat cell line was derived by transfecting human Jurkat T cells with the pSFFV-neo mammalian expression vector containing the human BCL-2 ORF insert and a neomycin-resistant gene. Has been for models of measuring efferocytosis.</td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermofisher</td>
			<td>AMQAX1000</td>
			<td>It is a benchtop assay platform equipped with state-of-the-art optics, full autofocus, and image analysis software for rapid assessment of cells in suspension. Very easy to use.</td>
		</tr>
		<tr>
			<td>Cytospin 4 Cytocentrifuge</td>
			<td>Thermofisher</td>
			<td>A78300003</td>
			<td>Provides economical thin-layer preparations from any liquid matrix, especially hypocellular fluids such as bronchoalveolar lavage fluid.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Thermofisher</td>
			<td>16140071</td>
			<td>Provides Nutrients to cultured cells for them to grow. It is standard for cell culture.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma/Aldrich</td>
			<td>P0781-100ML</td>
			<td>Penicillin-Streptomycin is the most commonly used antibiotic solution for culture of mammalian cells. Additionally it is used to maintain sterile conditions during cell culture.</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement</td>
			<td>Thermofisher</td>
			<td>61870036</td>
			<td>RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) was originally developed to culture human leukemic cells in suspension and as a monolayer. RPMI 1640 medium has since been found suitable for a variety of mammalian cells, including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas. Helps grow Jurkat T cells fast and efficiently.</td>
		</tr>
		<tr>
			<td>Stratagene UV Stratalinker 1800 UV Crosslinker</td>
			<td>Cambridge Scientific</td>
			<td>16659</td>
			<td>The Stratalinker UV crosslinker is designed to induce apoptosis, crosslink DNA or RNA to nylon, nitrocellulose, or nylon-reinforced nitrocellulose membranes.</td>
		</tr>
	</tbody>
</table>

Source: Hodge, M. X., et al. <a href="https://app.jove.com/v/60109">In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure.</a> J. Vis. Exp. (2019).
This video demonstrates a method to induce apoptosis in the Jurkat cells &#8212; a human T lymphocyte leukemia cell line. The cells are exposed to ultraviolet C (UVC) radiation, which causes extensive DNA damage and leads to apoptotic cell death.

1. Preparation of Jurkat T cell line (Day 2)
NOTE: All procedures should be conducted in a class II biological safety cabinet.

	Culture Jurkat T cells in ...

Take a suspension of Jurkat cells &#8212; T lymphocyte leukemia cells.
Mix an aliquot with a cell viability dye and ensure an adequate number of living cells.
Plate the cells in a culture dish and apply ultraviolet C or UVC, radiation for the required duration.
UVC penetrates the cells and is absorbed by DNA, forming pyrimidine photoproducts.
Post-irradiation, incubate the cells. Extensive UVC-mediated DNA damage initiates the intrinsic apoptosis pathway, activating pro-apoptotic proteins on the outer mitochondrial membrane.
Activated proteins oligomerize, forming membrane pores and releasing cytochrome&#160; C from the intermembrane space into the cytoplasm.
Cytochrome C complexes with the apoptotic protease activating factor-1, forming an apoptosome complex. The apoptosome activates caspases, causing apoptosis.
Introduce fluorophore-labeled annexin V to label phosphatidylserine on the membrane, an apoptotic cell marker, and add propidium iodide that enters through the damaged membrane to label the DNA.
Using flow cytometry, determine the annexin V-labeled early apoptotic cells and dual-stained late apoptotic cells.

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The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

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Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

高纯度凋亡体的检测与分离

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

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Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

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An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells

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An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells