Name: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice (Video) | JoVE Journal
Uploaded: 2011-04-03T14:00:00.000+00:00
Duration: 17 min 59 s
Description: Learn to establish thymic lymphoma T-cell lines from Atm-/- and p53-/- mice using this detailed protocol. This method enables cancer research and therapeutic target exploration.

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5 minutes

The authors wish to thank Dr. Boris Reizis from the Department of Microbiology and Immunology at Columbia University for helpful discussions. We also wish to thank Dr. Margaret Bynoe, Dr. Rodman Getchell and Deequa Mahamed from the Department of Microbiology and Immunology at the College of Veterinary Medicine, Cornell University for technical assistance with flow cytometry and Lu Huang for assistance with video editing. The authors also wish to thank the members of Duhamel and Weiss laboratories for their critical review of the manuscript and video production, and Stephanie Yazinski from the Weiss laboratory for breeding and genotyping p53-/- mice. Experiments on animals were performed in accordance with the guidelines and regulations set forth by Cornell University Institutional Animal Care and Use Committee. This research was supported in part by funds provided by the US Department of Agriculture, Cooperative State Research, Education, and Extension Service, Animal Health and Disease Research Program (to G.E.D. and R.S.W.) and NIH grants R03 HD058220 and R01CA108773 (to R.S.W). The video was produced using in-house facilities by the personnel from the Duhamel &amp; Weiss laboratories.

1. Dissection of Tumor
<ol>
	<li>For tumor dissection, a clean paper towel or disposable surgical drape is placed on the dissection pad and sprayed with 70% ethanol.</li>
	<li>Mice for this protocol are routinely euthanized with CO2.Place the euthanized mouse on the dissection pad and spray both sides of the animal with ethanol. The mouse is held in place onto the dissection pad by using four or more push pins inserted through the legs and sprayed thoroughly with 70% ethanol.</li>
	<li>The skin over the midline is cut from the pubis to the base of the head by using sterile scissors, while avoiding the underlying muscles and penetrating into the body cavities.</li>
	<li>Open the abdominal cavity with a new pair of sterile scissors and forceps being careful to not damage the internal organs.</li>
	<li>Open the thoracic cavity by dissecting and deflecting the rib cage to reveal the tumor, without damaging blood vessels.</li>
	<li>Separate the tumor from other organs, and transfer a piece approximately 40-50% of the total tumor (approximately up to 1.5 X 0.8 X 0.8 cm) into cold PBS kept on ice.</li>
	<li>The remaining portion of the tumor can be saved for other assays and a complete necropsy can be performed at this time.</li>
</ol>
2. Culturing Lymphoma Cells
<ol>
	<li>Spray the tube containing the dissected tumor with 70% ethanol and transfer to a biosafety cabinet.</li>
	<li>Add 10 mL of culture medium to a sterile 150 mm2 Petri dish.</li>
	<li>Transfer the piece of tumor into the Petri dish by gentle suction with a pipette.</li>
	<li>Bend two sterile 18 gauge needle facing the beveled side out.</li>
	<li>Dissociate the tumor using the bent needles.</li>
	<li>Swirl the dish, tap gently and place at an angle to collect the cell suspension to a side, while avoiding large pieces of tumor.</li>
	<li>Aspirate the cell suspension and transfer into a 50 mL conical tube while avoiding large pieces of tumor.</li>
	<li>Wash the plate with 10 mL of medium and transfer into the same tube while avoiding large pieces of tumor.</li>
	<li>Mix the cell suspension composed of lymphocytes and stromal cells gently and transfer 5 mL into a T-25 tissue culture flask and 10 mL into a T-75 tissue culture flask. Gently swirl the flasks.</li>
	<li>Use the remaining cell suspension to make 2, 4, 10,100 and 1000 fold dilutions in 2.5 mL final volume (culturing cells at different densities using several types of culture vessels maximizes the likelihood of establishing a T-cell line).</li>
	<li>Plate 2.0 mL of each dilution into individual wells of a 6-well plate. Gently swirl the plate.</li>
	<li>Incubate the cells for 48 h in humidified CO2 atmosphere in air.</li>
</ol>
3. Feeding Lymphoma Cells
<ol>
	<li>After 48 h of incubation, add 5 mL of medium to the T-25 flask and 10 mL of medium to the T-75 flask along the wall with minimal disturbance of the cells.</li>
	<li>Add 2 mL of medium to each well of the 6-well plate along the wall with minimal disturbance of the cells.</li>
	<li>Incubate the cells for an additional 72 h.</li>
</ol>
4. Passaging Lymphoma Cells
<ol>
<li>Initial passage
<ol>
<li>At the end of the fifth day of incubation, the cells are passaged at a 1:1 ratio, while retaining the original flasks and plates (maintaining the cell density at a concentration of approximately 3x106/ mL during the first passages promotes successful establishment of a continuous cell line).
<ol class="lalpha">
<li>For the T-25 flask, transfer 5 mL of cell suspension into a new flask containing 5 mL of medium. Add 5 mL of medium into the original flask.</li>
<li>For the T-75 flask, transfer 10 mL of cell suspension into a new flask containing 10 mL of medium. Add 10 mL of medium into the original flask.</li>
<li>For the 6-well plate, transfer 2 mL of cell suspension from each well into corresponding wells of a new plate containing 2 mL of medium in each well. Add 2 mL of medium into each well of the original wells.</li>
</ol>
</li>
<li>Incubate all flasks and plates for an additional 72 h. In our experience most cell lines are established from these passage one cultures (the presence of loosely adherent suspension cell aggregates over the attached stromal cells is an early indication of an established cell line).</li>
<li>For the cultures with fewer or no loosely adherent suspension cell aggregates, feed the flasks or plates by carefully replacing approximately 50% of the medium every 72 h. To avoid removing cells from the original culture, culture vessels are left standing and undisturbed for at least 5 min to let the cells sediment at the bottom by gravity before gentle aspiration of the supernatant medium.</li>
</ol>
</li>
<li>Maintaining established cell lines
<ol>
<li>Established cell lines are passaged into new flasks and plates. For T-25 and T-75 flasks, add 5 mL of the original or passage one suspension into 5 mL of medium. For 6-well plates, add 3 mL of the original or passage one cell suspension into 7 mL of medium in a T-25 flask. Feed and retain the original or passage one culture by replacing the volume removed with fresh culture medium.</li>
<li>Passage established cell lines into new T-25 flasks. The cell lines have a doubling time of 18-24 h. The cultures are routinely maintained at a density of 1-3x106/ mL and passaged every 3 days. Add the required volume of previous passage culture to new medium to make up to 10 mL. Feed and retain the original culture.</li>
<li>After a few passages, the non-adherent cells will self sustain without the adherent stromal cells and a smaller number of adherent cells are carried over with each passage. Beyond this point, the cultures can be maintained in non-tissue culture treated T-25 flasks.</li>
</ol>
</li>
</ol>
5. Freezing and Recovering Lymphoma Cell Lines
<ol>
	<li>The cell lines are frozen in freshly prepared RPMI 1640 medium containing 20% FBS and 10% DMSO.</li>
	<li>Cells from a 48 h old culture in T-25 flask (approximately 1.5x106 cells/ mL) are centrifuged at 1500 rpm for 5 min at room temperature. After discarding the supernatant, the cells are re-suspended in 2 mL of cold freezing medium. Two 1.0 mL volumes of cell suspension from each flask are placed in freezing vials, immediately frozen at -80°C, and transferred to liquid nitrogen the following day.</li>
	<li>Cells are recovered from liquid nitrogen storage by quick thawing, and resuspending the contents in 10 mL of fresh culture medium. Alternatively, freshly thawed cells are washed once in warm culture medium to remove the DMSO before resuspending in 10 mL of fresh culture medium. The cells are cultured for at least 72 h before subsequent passage.</li>
</ol>
6. Representative Results
An Atm-/- T-cell line developed using this protocol and designated DWJ-Atm-1 was characterized by staining with anti-CD3-FITC, anti-CD4-APC and anti-CD8-PE and flow cytometry analysis (Figure1). More than 90% of the T-cells were CD3, CD4 and CD8 triple-positive.
<img src="/files/ftp_upload/2598/2598fig1.jpg" alt="Flow cytometry dot plot and table; CD markers analysis; CD8-PE vs. CD4-APC; cell surface expression." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Characterization of a T-cell line developed using this protocol.
The cell surface markers of an established Atm-/- cell line designated DWJ-Atm-1 were characterized by staining with anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE and flow cytometry analysis. Briefly, 1X106 fresh cells were washed once in cold PBS (1500 rpm/5 min), and incubated at 4°C for 10 min with 200 μL of antibody cocktail (each antibody at 1:200 dilution in PBS with 1% BSA. Cells can be examined immediately while fresh or fixed for later analysis. Fixed cells are prepared by adding neutral buffered formalin at a 4% (vol/vol) final concentration and incubated at 4°C for 30 min, harvested by centrifugation (1500 rpm/5 min) and resuspended in 200 μL of PBS with 1% BSA. A total of 1 X 104 cells were analyzed by using a BD FACS Calibur analyzer and CellQuest software (BD Biosciences, San Jose, CA).

<ol><li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atm-deficient+mice%3A+a+paradigm+of+ataxia+telangiectasia%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li>
<li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li>
<li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+Atm-%2F-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone%5BTitle%5D)+AND+%22Cancer+Chemother+Pharmacol%22%5BJournal%5D)">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li>
<li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice%5BTitle%5D)+AND+%22Free+Radic+Biol+Med%22%5BJournal%5D)">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li>
<li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+spectrum+analysis+in+p53-mutant+mice%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li>
<li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL%2FLMO1+double+transgenic+mice%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li>
<li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li>
<li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li>
</ol>

No conflicts of interest declared.

In this protocol, we provide detailed procedures to establish murine T-cell lines from two different mouse genotypes; Atm-/- [FVB/N-Atmtm1Led/J] and p53-/- [129/S6-Trp53tm1Tyj/J]. By using this protocol, a total of 6 (out of 7 attempts) Atm-/- and three (out of 5 attempts) p53-/- murine T-cell lines have been established in our laboratory. Representative data demonstrating that cell line DWJ-Atm-1, an<e...

<table><tbody><tr><td colspan="4">&lt;ul&gt;&lt;li&gt;Ice bucket&lt;/li&gt;&lt;li&gt;70% ethanol&lt;/li&gt;&lt;li&gt;Dissection pad consisting of foam block covered with aluminum foil and pins&lt;/li&gt;&lt;li&gt;Two sets of small sterile scissors and forceps&lt;/li&gt;&lt;li&gt;Sterile 150 mm&lt;sup&gt;2&lt;/sup&gt; Petri dish (Thermo Fisher Scientific, Waltham, MA; cat. #08-757-13)&lt;/li&gt;&lt;li&gt;Sterile 18 gauge needles&lt;/li&gt;&lt;li&gt;50 mL conical tubes (Corning Inc., Corning NY; cat # 430829)&lt;/li&gt;&lt;li&gt;10% buffered formalin&lt;/li&gt;&lt;li&gt;Tissue culture flasks &lt;ul&gt;&lt;li&gt;T-25 (TPP, Trasadingen, Switzerland; cat # 90025)&lt;/li&gt;&lt;li&gt;T-75 (TPP, Trasadingen, Switzerland; cat # 90075)&lt;/li&gt;&lt;/ul&gt;&lt;/li&gt;&lt;li&gt;6-well plates (Corning Inc., Corning NY; cat # 3506)&lt;/li&gt;&lt;li&gt;Sterile phosphate buffered saline pH 7.2 (PBS)&lt;/li&gt;&lt;li&gt;Culture medium &lt;ul&gt;&lt;li&gt;RPMI 1640 with 25 mM HEPES, 200 mM L-glutamine (Lonza, Walkersville, MD; cat#12-115F) supplemented with the following:&lt;ul&gt;&lt;li&gt;10% heat inactivated (56&amp;#x00B0;C, 30 min) fetal bovine serum (FBS; Hyclone, Logan, UT; cat# SH30088.03)&lt;/li&gt;&lt;li&gt;1% penicillin and streptomycin (Mediatech, Manassas,VA; cat# 30-002-CI)&lt;/li&gt;&lt;li&gt;1% nonessential amino acids (Mediatech; cat#25-025-CI)&lt;/li&gt;&lt;li&gt;55 mM 2&amp;#946;-mercapt&amp;#8211;thanol (Sigma, St Louis, MO; cat#M752)&lt;/li&gt;&lt;/ul&gt;&lt;/li&gt;&lt;/ul&gt;&lt;/li&gt;&lt;li&gt;Dimethyl sulfoxide (DMSO; Calbiochem, La Jolla, CA; cat # 317275)&lt;/li&gt;&lt;li&gt;Antibodies (eBioScience, San Diego, CA)&lt;ul&gt;&lt;li&gt;anti-CD3-FITC (cat. #11-0031-85)&lt;/li&gt;&lt;li&gt;anti-CD4-APC (cat. #17-0041-83)&lt;/li&gt;&lt;li&gt;anti-CD8-PE (cat.#12-081-85)&lt;/li&gt;&lt;/ul&gt;&lt;/li&gt;&lt;li&gt;Bovine serum albumin (BSA; Life Technologies, Grand Island, NY; cat #. 11018-017)&lt;/li&gt;&lt;li&gt;Concanavalin A (Sigma cat# C5275)&lt;/li&gt;&lt;li&gt;1.8 mL freezing vials (Nunc, Roskilde, Denmark; cat# 368632)&lt;/li&gt;&lt;/ul&gt;</td></tr></tbody></table>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

In this video, we demonstrate a protocol to establish mouse thymic lymphoma. Cell lines. Tumors were harvested for mice age three to six months at the earliest syndication of visible tumors.Based on the observation of hunched posture, labored breathing, poor grooming, and wasting, we have successfully established several ATM null and P 53 null thymic lymphoma cell lines Using this protocol. In part one, we describe how to harvest the thymic lymphoma from a tumor bearing.Mouse. Mice for this protocol are routinely euthanized with carbon dioxide.Place a clean paper towel on the dissection pad and spray with 70%ethanol. Place the euthanized mouse on the dissection pad. Spray both sides of the animal with ethanol.Pin the mouse onto the dissection pad and spray thoroughly with 70%ethanol. Open the skin using sterile scissors and forceps while taking care not to penetrate into body cavities. Open the abdominal cavity with a new pair of sterile instruments.Take care not to damage any organs. Open the thoracic cavity by dissecting and deflecting the rib cage to reveal the tumor without damaging any blood vessels. This mouse has a well-developed tumor occupying the entire thoracic cavity.Separate the tumor from other organs and excise a piece approximately 40 to 50%of the volume of the tumor. Transfer the Piece of tumor into cold PBS. Take another piece of the tumor and fix it in 10%formin.For histology, you may save the remaining portion of the tumor. For other assays, you additionally may examine and fix other organs at this time. In this section, we describe how to culture the thymic lymphoma cells from harvested tumors.Braid the tube containing the dissected tumor with 70%ethanol and transfer it into a tissue culture hood. Add 10 milliliters of culture medium to the sterile Petri dish. Transfer the piece of tumor into Petri dish with gentle suction from the pipette bend two sterile 18 gauge needles facing the Beveled side out.Dissociate the piece of tumor using the bent needles. Swirl the dish tap gently and place at an angle to collect the cell suspension to a side while avoiding large tumor pieces. Aspirate the cell suspension and transfer into a 50 milliliter conical tube.While avoiding large tumor pieces. Wash the plate with 10 milliliters of medium and transfer into the same tube. Avoid large tissue Particles.Mix the cell suspension gently and transfer five milliliters into a T 25 tissue culture flask. Transfer 10 milliliters into a T 75 tissue culture flask. Use the remaining self suspension to make 2 4 10 101, 000 Fold dilution Plate two milliliters of each dilution into individual wells of a six Well plate Gently swirl the plate, incubate the cells for 48 hours.In this section, we demonstrate how to feed the cultures until they're ready to passage. After 48 hours of incubation, add five milliliters of medium into the T 25 flask. Add 10 milliliters of medium into the T 75 flask along the wall with minimal disturbance to the cells.Add two milliliters of medium to each well. Of the six well plate along the wall incubate the cells for 72 hours. In this section, we will describe the procedures for packaging and maintaining the established cell lines.At the end of five days, passage the cells at a one-to-one ratio and retain the original flask For the T 25 flask. Transfer five milliliters of cell suspension into a new flask containing five milliliters of Medium. Add five milliliters of medium into the original flask For the T 75 flask.Transfer 10 milliliters of cell suspension into a new flask containing 10 milliliters of medium. Add 10 milliliters of medium into the original flask. For the six well plate transfer two milliliters of cell suspension from each well into the corresponding well of a new plate containing two milliliters of medium In each well.Add Two milliliters of medium into each well of the original plate. Incubate the cells for 72 hours. In our experience, most cell lines were established from these passage.One cultures at the end of 72 hours. Observe the flasks and plates for the presence of loosely adherent suspension. Cell aggregates over the attached stromal cells.This is an early indication of an established cell line for the cultures with fewer loosely adherent suspension. Cell aggregates feed the flasks or plates by replacing 50%media every 72 hours taking care not to remove cells passage. Establish cell lines into new T 25 flask.The cell lines have a doubling time of 18 to 24 hours. The cultures are routinely maintained at a density of one to 3 million cells per milliliter and passage every three days. Add the required volume of previous passage culture to new medium to make up to 10 milliliters, Feed and retain the original culture.After a few passages, the non-adherent cells will self sustain without the adherent stromal cells and a smaller number of adherent cells will be carried over with each passage. In our experience, most cell lines became established between passage two and eight. Beyond this point, the cultures can be maintained in non tissue culture treated T 25 flasks.The established cell lines were routinely characterized for cell surface markers by flow cytometry for most of the cell lines, more than 80%of the population with CD three, CD four, and CD eight positive T cells. In the final section, we describe how to prepare freezer stocks for liquid nitrogen storage and how to recover the cell lines. The cell lines are routinely frozen in culture medium with 20%fetal bovine serum and 10%dimethyl sulfoxide two one milliliter.Freezing vials are prepared from each 48 hour old T 25 flask. Cells are centrifuge at 1500 RPM for five minutes of room temperature. Supernat is discarded and cells are resuspended in cold freezer, medium, immediately frozen at negative 80 degrees Celsius and transferred to liquid nitrogen.The following day, cells are recovered from liquid nitrogen storage by quickly thawing a vial Resus suspending the contents in 10 milliliters of prewarm culture, media, and culturing for 72 hours before ing several thymic lymphoma cell lines are in culture over one year after being established under this protocol in our laboratory. We wish you good luck in your experiments.

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Learn to establish thymic lymphoma T-cell lines from Atm-/- and p53-/- mice using this detailed protocol. This method enables cancer research and therapeutic target exploration.

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