This research was funded by the NIH grants K08 AI062732, K12 HD001401, and 1UL1RR024139-02 and a Child Health Research Grant from the Charles H. Hood
Foundation to S.B.-M. and by the Research Foundation at the State University of New York at Stony Brook.

1. EBV stock preparation
<ol>
	<li>Subculture exponentially growing B95-8 cells (ATCC # CRL 1612; 13) at 3 x 105 cells/ml in a 75cm2 tissue culture flask using sterile technique. Cells are grown in complete RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), Penicillin/Streptomycin at 100U/ml and 100 &mu;g/ml, respectively, and Amphotericin B at 0.5 &mu;g/ml at 37&deg;C in the presence of 5% CO2.</li>
	<li>Forty-eight hours later, resuspend cells in fresh complete RPMI 1640 at 1 x 106 cells/ml. To induce virus production, stimulate cells with 20ng/ml tetradecanoyl phorbol acetate (TPA) for 1 hour in a standard CO2 incubator. Wash cells three times with RPMI 1640 to remove TPA.</li>
	<li>Resuspend cells in the original volume of complete RPMI 1640 (from 1.2) and place the flask in CO2 incubator for 96 hours. This method has been shown to produce high titers of infectious virus particles6.</li>
	<li>Centrifuge at 600 x g for 10 min at 4&deg;C to separate EBV-containing culture supernatant from cells. Filter supernatant through a 0.45-micron filter, aliquot, and store at -70&deg;C for over a year. 
	An alternative is to obtain the supernatant from B95-8 cells containing EBV from the ATCC (VR-1492) and use at the dilution recommended by the ATCC.</li>
</ol>
2. Isolation of peripheral blood mononuclear cells (PBMC)
<ol>
	<li>Draw 10 ml blood from donor into a heparinized syringe or a heparinized blood tube. Dilute blood with 20 ml PBS at room temperature in a 50 ml conical tube.</li>
	<li>Underlay diluted blood with 15 ml Ficoll Hypaque lymphocyte separation medium. Centrifuge at 225 x g without brake at room temperature for 30 minutes.</li>
	<li>Remove buffy coat and transfer into a new 50 ml conical tube. Raise volume to 50 ml with PBS and spin at 600 x g at room temperature for 10 minutes.</li>
	<li>Pour off supernatant. Wash cells by resuspending pellet in 50 ml PBS and spinning at 600 x g at room temperature for 10 minutes. Wash twice more in a similar manner.</li>
	<li>Resuspend washed cells in 1 ml of complete RPMI. Use 5 &mu;L of cells to prepare a 1/10 dilution in Trypan blue. Count live cells using a hemocytometer.</li>
	<li>Adjust the volume using complete RPMI in a 25 cm2 tissue culture flask to obtain a cell concentration of 2 x 106/ml.</li>
</ol>
3. Infection with EBV
<ol>
	<li>Add FK506 (AG Scientific) to the cell suspension from 2.6 to a final concentration of 20 nM. Place flask in CO2 incubator at 37&deg;C for one hour.</li>
	<li>Rapidly thaw an aliquot of EBV. Remove flask from incubator and add EBV to the cells at 1/10 dilution. Typically, this dilution provides an MOI of 50-100. Swirl flask to mix and place it upright in CO2 incubator at 37&deg;C.</li>
</ol>
Note that Step 4, performed to predict successful outcome, is an optional step. If Step 4 is to be performed, you will need to incubate an additional flask of un-infected PBMC at 2 x 106 cells/ml as control.
4. Identifying proliferating population of cells (Optional step)
<ol>
	<li>Prepare FACS buffer: PBS + 5% FBS. Store at 4&deg;C.</li>
	<li>Make the following antibody mixes in 50 &mu;L volume each for staining cells in Step 4.6. Antibody dilutions should be made in FACS buffer containing 1mg/ml of mouse IgG (Sigma) to inhibit non-specific binding by antibodies.
	<ol class="lroman">
		<li>single-stained with PE conjugated anti-CD23 antibody (1/50 dilution)</li>
		<li>single-stained with FITC conjugated anti-CD58 antibody (1/50 dilution)</li>
		<li>single-stained with PE-Cy5 conjugated anti-CD19 antibody (1/50 dilution)</li>
		<li>triple-stained for CD23, CD58, and CD19 (1/50 dilution each)</li>
		<li>single-stained with PE conjugated isotype control antibody matched to CD23-PE (at the same concentration as anti-CD23 antibody)</li>
		<li>single-stained with FITC conjugated isotype control antibody matched to CD58-FITC (at the same concentration as anti-CD58 antibody)</li>
		<li>single-stained with PE-Cy5 conjugated isotype control antibody matched to CD19-PE-Cy5 (at the same concentration as anti-CD19 antibody)</li>
		<li>triple-stained with all three isotype control antibodies.</li>
	</ol>
	Temporarily store the mixes at 4&deg;C in the dark.</li>
	<li>In day three post-exposure to EBV, gently swirl flask to obtain a more uniform cell suspension. Remove 2ml from flask into a 2ml Eppendorf tube. Spin tube in a microcentrifuge at 3000 rpm at room temperature for 3 minutes.</li>
	<li>Aspirate supernatant and resuspend cells in FACS buffer at 5 x 106 to 1 x 107cells/ml.</li>
	<li>Remove eight 50&mu;l aliquots into individual Eppendorf tubes or a 96-well V-bottom plate. Spin tubes or the plate at 1500 x g for 3 minutes.</li>
	<li>Discard supernatant and vortex tubes/plate. Resuspend cells in each tube/well in 50 &mu;L of FACS buffer containing appropriate antibody dilutions prepared in 4.2.</li>
	<li>Incubate tubes/plate on ice in the dark for 30 minutes. Centrifuge cells at 3000 rpm for 3 minutes, remove supernatant, and add 200 &mu;L of FACS buffer. Centrifuge cells again and remove supernatant to complete the wash. Repeat two more washes.</li>
	<li>Resuspend cells in 200 &mu;L FACS buffer and acquire data using a flow cytometer, acquiring 20,000 events/tube.</li>
	<li>Analyze data using WinMDI (freeware for FACS data analysis on PC). Gate on live cells using forward and side scatter profiles. Then, gate live cells for expression of CD19+ (B cell marker) after comparing with cells stained with isotype control antibody matched to anti-CD19 antibody. Plot CD19+ live B cells with fluorescence intensity for CD23 on x-axis and fluorescence intensity for CD58 on y-axis. Determine CD23+ and CD58+ cells by comparing to EBV-exposed cells stained with matched isotype control antibodies. Presence of CD23hiCD58+ cells in EBV-exposed culture (Figure 2C) predicts successful outgrowth of EBV-infected growth transformed cells. In contrast, CD23hiCD58+ cells do not emerge when cells are not exposed to EBV (Figure 2A) or exposed to a non-immortalizing strain of EBV (Figure 2B). CD23hiCD58+ cells have been experimentally demonstrated to undergo proliferation and subsequently establish LCL (14).</li>
</ol>
5. Expansion and cryopreservation of LCL
<ol>
	<li>Visualization of cells by light microscopy: By a week after EBV infection, clusters of cells are visible by light microscopy. Figure 3 shows an example of early microscopic clusters (Figure 3B) in a flask.</li>
	<li>As time progresses, microscopic clusters become larger such that clumps are visible macroscopically in the flask. Figure 3C shows larger clusters of cells in established LCL by light microscopy.</li>
	<li>Feeding cells: Double the volume of culture medium in the culture flask on day 12. Subsequently, expand culture by increasing its volume 2-3 fold using complete RPMI.</li>
	<li>Periodicity of feeding cells should be determined based on rate of cell growth. When the culture medium turns yellow, it is generally time to replace the media as above. Typically, this occurs once per week. However, some cell lines may need to be fed more or less frequently. Expand culture to 75-100 ml over the next few weeks.</li>
	<li>Cryopreservation: When freezing down cells, centrifuge cells at 600 x g for 10 minutes at room temperature. Remove the supernatant and resuspend cell pellet in cold freezing medium containing 90% FBS and 10% dimethylsulfoxide at 1 x 107 cells/ml. Place tube in liquid nitrogen.</li>
</ol>
6. Representative results:
Successful outcome is predicted by the presence of CD23hiCD58+ cells. Approximately 2-3% of live cells on day 3-4 after exposure to EBV should have this profile (Figure 2C). Other sub-populations of CD23+, CD23-, CD58+, and CD58- cells observed after exposure to EBV show minimal proliferation14. Un-infected cells (Figure 2A) and cells exposed to EBV from HH514-16 cells (Figure 2B), harboring a non-immortalizing strain of EBV, do not demonstrate CD23hiCD58+cells.
You may also visualize cells by light microscopy to monitor successful outcome: As mentioned earlier, within a week after EBV infection, small clusters of cells in the flask are visible by light microscopy (Figure 3B). As time progresses and cells develop into LCL, microscopic clusters become larger (Figure 3C) such that clumps are visible macroscopically in the flask.
<img alt="Figure 1" src="/files/ftp_upload/3321/3321fig1.jpg" /> 
Figure 1. Workflow for generation and cryopreservation of lymphoblastoid cell lines. Peripheral blood is centrifuged through a Ficoll gradient. PBMC present in the buffy coat of an established gradient are exposed to FK506 followed by addition of EBV. EBV-exposed cells are grown at 37&deg;C in the presence of 5% CO2 to establish and subsequently expand LCL for cryopreservation.
<img alt="Figure 2" src="/files/ftp_upload/3321/3321fig2.jpg" /> 
Figure 2. Identification of a sub-population of B cells expected to undergo proliferation after exposure to EBV. Un-infected PBMC (A) or PBMC exposed to EBV derived from HH514-16 cells (B) or from B95-8 cells (C) in the presence of FK506 were harvested on day 3. Cells were stained with fluorochrome-conjugated antibodies directed against CD19, CD23, and CD58. After gating on live cells, un-infected (A) and EBV-exposed (B and C) CD19+ B cells were examined for expression of CD23 and CD58. A sub-population of B cells expressing CD58 and high levels of CD23 (CD23hiCD58+), observed only in the cells exposed to transformation competent EBV (derived from B95-8 cells), is depicted.
<img alt="Figure 3" src="/files/ftp_upload/3321/3321fig3.jpg" /> 
Figure 3. Visualization of cell clusters in EBV-infected cells. PBMC were treated with FK506 and infected with EBV. Un-infected (A) and EBV-exposed (B) cells were examined one week post-infection by phase contrast microscopy (10X magnification). Five-week old lymphoblastoid cell line is shown in C.

<ol>
	<li>Thorley-Lawson, D. A., Gross, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+the+Epstein-Barr+virus+and+the+origins+of+associated+lymphomas.">Persistence of the Epstein-Barr virus and the origins of associated lymphomas.</a> N. Engl. J. Med. 350, 1328-1337 (2004).</li><li>Kubuschok, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+spontaneous+Epstein-Barr+virus+lymphoblastoid+cell+lines+genetically+modified+to+express+tumor+antigen+as+cancer+vaccines:+mutated+p21+ras+oncogene+in+pancreatic+carcinoma+as+a+model.">Use of spontaneous Epstein-Barr virus lymphoblastoid cell lines genetically modified to express tumor antigen as cancer vaccines: mutated p21 ras oncogene in pancreatic carcinoma as a model.</a> Hum. Gene Ther. 13, 815-827 (2002).</li><li>Kuppers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+under+influence:+transformation+of+B+cells+by+Epstein-Barr+virus.">B cells under influence: transformation of B cells by Epstein-Barr virus.</a> Nat. Rev. Immunol. 3, 801-812 (2003).</li><li>Traggiai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+to+make+human+monoclonal+antibodies+from+memory+B+cells:+potent+neutralization+of+SARS+coronavirus.">An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.</a> Nat. Med. 10, 871-875 (2004).</li><li>Bernasconi, N., Traggiai, E., Lanzavecchia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+serological+memory+by+polyclonal+activation+of+human+memory+B+cells.">Maintenance of serological memory by polyclonal activation of human memory B cells.</a> Science. 298, 2199-2202 (2002).</li><li>Oh, H. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+rapid+establishment+of+Epstein-Barr+virus+immortalization+of+human+B+lymphocytes.">An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes.</a> Cell Prolif. 36, 191-197 (2003).</li><li>Ventura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+simple+method+for+the+Epstein-Barr+virus+transformation+of+lymphocytes+from+members+of+large+families+of+Reunion+Island.">Use of a simple method for the Epstein-Barr virus transformation of lymphocytes from members of large families of Reunion Island.</a> Hum. Hered. 38, 36-43 (1988).</li><li>Henderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+transformation+of+lymphocytes+by+Epstein-Barr+virus.">Efficiency of transformation of lymphocytes by Epstein-Barr virus.</a> Virology. 76, 152-163 (1977).</li><li>Bird, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Epstein-Barr+virus+activation+of+human+B+lymphocytes.">Characteristics of Epstein-Barr virus activation of human B lymphocytes.</a> J. Exp. Med. 154, 832-839 (1981).</li><li>Neitzel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+routine+method+for+the+establishment+of+permanent+growing+lymphoblastoid+cell+lines.">A routine method for the establishment of permanent growing lymphoblastoid cell lines.</a> Hum. Genet. 73, 320-326 (1986).</li><li>Pelloquin, F., Lamelin, J. P., Lenoir, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+lymphocytes+immortalization+by+Epstein-Barr+virus+in+the+presence+of+cyclosporin+A.">Human B lymphocytes immortalization by Epstein-Barr virus in the presence of cyclosporin A.</a> In Vitro Cell Dev. Biol. 22, 689-694 (1986).</li><li>Pressman, S., Rotter, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+transformation+of+cryopreserved+lymphocytes:+prolonged+experience+with+technique.">Epstein-Barr virus transformation of cryopreserved lymphocytes: prolonged experience with technique.</a> Am. J. Hum. Genet. 49, 467-467 (1991).</li><li>Miller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus:+Transformation,+cytopathic+changes,+and+viral+antigens+in+squirrel+monkey+and+marmoset+leukocytes.">Epstein-Barr virus: Transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes.</a> Proc. Nat. Acad. Sci. 69, 383-387 (1972).</li><li>Megyola, C., Ye, J., Bhaduri-McIntosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+sub-population+of+B+cells+that+proliferates+after+infection+with+Epstein-Barr+virus.">Identification of a sub-population of B cells that proliferates after infection with Epstein-Barr virus.</a> Virol. J. 8, 84-84 (2011).</li><li>Tosato, G., Cohen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Epstein-Barr+virus+(EBV)-immortalized+B+cell+lines.">Generation of Epstein-Barr virus (EBV)-immortalized B cell lines.</a> Current Protocols of Immunology. Chapter 7, 22-22 (1991).</li></ol>

No conflicts of interest declared.

The method described in this paper generates LCL from donor peripheral blood with rapid immortalization and cryopreservation times. Through the use of FK506, a T cell immunosuppressant, and high titers of infectious virus we are able to promote proliferation of EBV-infected B cells from peripheral blood mononuclear cells. These interventions make the described method more efficient, resulting in rapid expansion of cells for subsequent experiments. 
Traditionally, growth transformation has been monitored by visualization of clusters of cells by light microscopy about a week after exposure to EBV6, 15. However, clustering of cells is not a specific indicator of EBV-mediated growth transformation. We have previously demonstrated consistent identification of the proliferating cell population via flow cytometry14, providing an accurate and specific method to determine successful outcome as early as three days after exposure of B cells to EBV.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Fisher Bioreagents</td>
			<td>BP 928-250</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide </td>
			<td>Sigma</td>
			<td>D 2650</td>
		</tr>
		<tr>
			<td>Fetal bovine serum, heat-inactivated</td>
			<td>Sigma</td>
			<td>F 4135</td>
		</tr>
		<tr>
			<td>Ficoll Hypaque lymphocyte separation media</td>
			<td>Mediatech</td>
			<td>25-072</td>
		</tr>
		<tr>
			<td>FK506</td>
			<td>A.G Scientific</td>
			<td>F 1030</td>
		</tr>
		<tr>
			<td>IgG from mouse serum</td>
			<td>Sigma</td>
			<td>I 8765</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD23 conjugated to PE</td>
			<td>BD Pharmingen</td>
			<td>555711</td>
		</tr>
		<tr>
			<td>Mouse isotype IgG1 conjugated to PE</td>
			<td>BD Pharmingen</td>
			<td>554680</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD58 conjugated to FITC</td>
			<td>Pierce Thermo Scientific</td>
			<td>MA1-82159</td>
		</tr>
		<tr>
			<td>Mouse isotype IgG1 conjugated to FITC</td>
			<td>BD Pharmingen</td>
			<td>550616</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD19 conjugated to PE-Cy5</td>
			<td>BD Pharmingen</td>
			<td>555414</td>
		</tr>
		<tr>
			<td>Mouse isotype IgG1 conjugated to PE-Cy5</td>
			<td>Dako</td>
			<td>X 0955</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Sigma	</td>
			<td>R 8758</td>
		</tr>
		<tr>
			<td>Tetradecanoyl phorbol acetate (TPA)	</td>
			<td>Calbiochem</td>
			<td>407952</td>
		</tr>
		<tr>
			<td>FACS Buffer (1X Phosphate Buffered Saline + 5% Fetal Bovine Serum)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

establishment of epstein-barr virus growth-transformed lymphoblastoid cell lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

Watch this Scientific Journal Video about Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines at JoVE.com

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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Immunology and Infection

39.8K Views.  State University of New York at Stony Brook. We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Video: Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

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.

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

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 ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...