This work was supported by the key projects of Chinese Academy of Sciences (KFZD-SW-205), strategic biological resources technology support system of Chinese Academy of Sciences (CZBZX-1).

This study was approved by the Ethics Committee of the Institute of Genetics and Developmental Biology, and the protocol follows the institutional guidelines for human welfare.
1. Preparation of EB Virus
<ol>
	<li>On day 1, thaw B95-8 cells and culture in 6 mL complete RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 &#181;g/mL streptomycin, 100 U/mL penicillin) in a T25 culture flask. Place the culture flask in 5% CO2 incubator at 37 &#176;C.</li>
	<li>On day 2, observe the cell morphology under a microscope. Cells are in good condition when they are bright and round. Add 10 mL complete RPMI 1640 medium.</li>
	<li>On day 4, add 10 mL fresh complete RPMI 1640 medium, and then transfer to a T75 culture flask.</li>
	<li>On day 8, add 20 mL complete RPMI 1640 medium.</li>
	<li>Maintain the cells for 8 days without adding fresh medium, i.e., starvation.</li>
	<li>On day 16, observe cells observed under the microscope. Collect the cells in a 15 mL centrifuge tube, 12 mL/tube.</li>
	<li>Cryo-preserve at -80 &#176;C for 1 h, and then rapidly thaw at 37 &#176;C. Repeat 3 times until the virus particles are released from the cells.</li>
	<li>Centrifuge at 240 x g for 10 min at room temperature.</li>
	<li>Filter the supernatant through a 0.22 &#181;m filter, aliquot, 12 mL/tube, and store at -80 &#176;C.</li>
</ol>
2. Treatment of Whole Blood with Red Blood Cell Lysis Buffer
<ol>
	<li>Add 0.5 mL whole blood anticoagulated by EDTA&#903;K2 to a 15 mL tube.</li>
	<li>Put 1 mL red blood cell lysis buffer (filtered by 0.22 &#181;m filter and thawed to room temperature before use) to the whole blood and mix well. Place the mixture at room temperature for 4 min. Then add 5 - 7 mL 1x PBS quickly to the stop reaction.</li>
	<li>Centrifuge at 240 x g for 5 min at room temperature. White blood cells will precipitate at the bottom of the tube.</li>
	<li>Discard the supernatant, and wash the precipitation by adding 7 mL 1640 basic medium. Centrifuge for 5 min at 240 x g at room temperature. Repeat twice.</li>
	<li>Resuspend the cell pellet in 300 &#181;L transformation medium (RPMI 1640 medium, containing 20% fetal bovine serum and 10 &#181;g/mL phytohemagglutinin (PHA)).</li>
</ol>
3. EBV Infection
<ol>
	<li>Add 60 &#181;L of 20 &#181;g/mL cyclosporin A (CsA) to the cell suspension.</li>
	<li>Rapidly thaw an aliquot of EBV at 37 &#176;C. Add 240 &#181;L EBV to the cell suspension.</li>
	<li>Mix well and place the cell suspension to a 96-well plate, 200 &#181;L per well. To avoid evaporation, add 200 &#181;L 1x PBS to every well around of the 96-well plate. Place the plate in a 5% CO2 incubator at 37 &#176;C.</li>
</ol>
4. Expansion and Cryopreservation of B-LCLs
<ol>
	<li>Change half of the medium once per week for the first month after EBV infection. Only a thick layer of cell fragments is visible under microscope at day 7 (Figure 2). After one month of EBV infection, lymphoblastoid cell clusters are visible under a thick layer of cell fragments under the microscope (Figure 2).</li>
	<li>Change half of the medium every 3 days until the cell clusters are clearly visible under the microscope (Figure 2). Typically, this process takes 10 to 20 days.</li>
	<li>After mixing, pipet the cells and transfer to the 24-well plate. Double the volume of culture medium with complete RPMI 1640 while it turns yellow. This process depends on the rate of cell growth.</li>
	<li>Subsequently, transfer the cells to T25 culture flask. Double the volume of culture medium as it turns yellow until the volume of medium expands to 10 - 15 mL.</li>
	<li>Change medium 24 h before freezing. Centrifuge cell suspension for 5 min at 240 x g. Count cells with a cell counter and freeze at a density of 5 - 10 x 106/mL in frozen stock solution containing 90% FBS and 10% dimethylsulfoxide (DMSO). Freeze at a rate of 1 &#176;C per min to -80 &#176;C and then transfer directly into liquid nitrogen.</li>
</ol>
5. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide (MTT) Assay17
<ol>
	<li>Prepare a B-LCLs single cell suspension with the 1640 medium containing 10% fetal bovine serum. Seed 3 x 104 cells per well onto 96-well plate, 100 &#181;L per well.</li>
	<li>Place the plate in 5% CO2 incubator at 37 &#176;C for 12 - 16 h.</li>
	<li>Add MTT (5 mg/mL) to wells in which cells are growing and keep away from light for 4 h at 37 &#176;C.</li>
	<li>Centrifuge cell plate for 10 min at 168 x g.</li>
	<li>Remove supernatant and add 100 &#181;L DMSO per well. 
	NOTE: Be careful. Do not touch the needle-shaped dye crystals.</li>
	<li>Shake the plate for 10 min at low speed to fully dissolve the crystals.</li>
	<li>Measure absorption at 570 nm by using a microplate spectrophotometer.</li>
</ol>

<ol>
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H., Quackenbush, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+lymphoblastoid+cell+lines+from+monozygotic+twins+discordant+in+severity+of+autism+reveals+differential+regulation+of+neurologically+relevant+genes.">Gene expression profiling of lymphoblastoid cell lines from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes.</a> BMC Genomics. 7, 118 (2006).</li><li>Toda, T., Sugimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Epstein-Barr+virus-transformed+B-lymphoblasts+and+the+proteome+database.">Proteome analysis of Epstein-Barr virus-transformed B-lymphoblasts and the proteome database.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 787 (1), 197-206 (2003).</li><li>Chiorazzi, N., Wasserman, R. L., Kunkel, H. G. <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+Epstein-Barr+virus-transformed+B+cell+lines+for+the+generation+of+immunoglobulin-producing+human+B+cell+hybridomas.">Use of Epstein-Barr virus-transformed B cell lines for the generation of immunoglobulin-producing human B cell hybridomas.</a> J Exp Med. 156 (3), 930-935 (1982).</li><li>Traggiai, E., et al. <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 (8), 871-875 (2004).</li><li>Hussain, T., Kotnis, A., Sarin, R., Mulherkar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+&amp;+characterization+of+lymphoblastoid+cell+lines+from+patients+with+multiple+primary+neoplasms+in+the+upper+aero-digestive+tract+&amp;+healthy+individuals.">Establishment &amp; characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract &amp; healthy individuals.</a> Indian J Med Res. 135 (6), 820-829 (2012).</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 (4), 320-326 (1986).</li><li>Oh, H. 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The authors declare that they have no competing financial interests.

We report the development of a novel method for immortalizing human B cells from whole blood by lysing red blood cells, and its cell viability of established B-LCLs was evaluated by the MTT assay. The results showed that the cell viability of B-LCLs established by the red blood cell lysis method is much higher than that of the density gradient separation method. The major advantage of the red blood cell lysis method is that it is simple and requires a small volume (as little as 0.5 mL) of blood for establishing a permanent cell line with a high success rate and high cell viability.
Only one simple lysis step is needed before EBV infection. Without previous purification of the lymphocytes, cell loss and cellular damage are avoided. Another obstacle of the density gradient separation method is hemolysis. Processing the blood after long term storage at inappropriate temperatures and shaking during transportation may lead to hemolysis, which will greatly influence the isolation of lymphocytes. Other studies showed that use of red blood cell (RBC) lysis resulted in higher numbers of viable PBMC than use of the density gradient separation method18 and lymphocyte subtypes were not influenced substantially19. Compared with the density gradient separation method, which is costly and cumbersome, the red blood cell lysis method is much easier, especially for treating a large number of the clinical samples and more practical for novices.
In the study, we have immortalized 24 samples with a 100% success rate by using the red blood cell lysis method. Compared with the 90-94% of success rate reported by cryopreserved lymphocytes and freshly isolated lymphocytes20 with 2 - 2.5 mL blood, this success rate is much higher. Meanwhile, we also transformed fresh (0.5 mL) or frozen (even 1 mL) blood as described in references14,15 without separation, and the efficiency of transformation is much lower than that reported16 (data not shown).
The present strategy not only improves immortalization efficiency significantly, but also reduces the volume of blood required to establish B-LCLs. Current technologies for EBV transformation use relatively large volumes of peripheral blood, approximately 2 - 2.5 mL blood20 to isolate lymphocytes while others require 10 mL or more10,21. This contrasts to the 0.5 mL of whole blood needed to obtain an immortalized cell line with the method described here. This method could be modified to successfully immortalize blood collected from finger pricks12, which could greatly facilitate preserve genetic resources from pediatric patients and the elderly.
In addition, the cell viability of B-LCLs established by the red blood cell lysis method are higher than that of the density gradient separation method. The red blood cell lysis method avoids the mechanical centrifugal force in the process of density gradient centrifugation, which causes non-repairable damage to the cells. It suggests that the red blood cell lysis method is more suitable for genetic and functional studies, which requires large numbers of cells as quickly as possible. Our method will decrease the time from culture initiation to cryopreservation, which will save resources and reduce labor.
A possible limitation of the red blood cell lysis method is the presence of contaminating debris derived from red blood cell lysates, which would block the clear observation of transformation in the first four weeks of EBV infection. However, this debris will be gradually removed with the medium changes. Another limitation is the loss of growing transformed cells, but this can be ameliorated by removing half of the supernatant in the first four weeks of transformation. Another possible modification to remove impurities is more frequent medium changes with greater volumes or washing the cell lines twice by 10 mL 1x PBS and then centrifuging at 168 x g for 5 min to remove impurities.
In summary, this protocol provides a simple strategy for establishing B-LCLs with high immortalization efficiency, reducing blood volume and saving time between initiation and cryopreservation. The cell lines developed in the present study provide a valuable and cost-effective source of biomaterials carry out various studies.

Resting B cells could be transformed by the Epstein-Barr virus (EBV) in vitro into actively proliferating B lymphoblastoid cell lines (B-LCLs). B-LCLs are similar to germ cells in differentiation and development, and the somatic mutation rate of B-LCLs was only 0.3%1, with negligible genetic and phenotypic alteration. Therefore, B-LCLs, as surrogates for peripheral blood mononuclear cells (PBMCs), have substantially accelerated the progress of genomics, transcriptomics and proteomics study of EBV associated diseases2,3. Moreover, B-LCLs also have important application in screening monoclonal antibodies4,5. 
Several methods have been developed for the immortalization of B lymphocytes by EBV. The most major common procedures include the isolation of B lymphocytes and the use of B95-8 cell lines to produce infectious EBV. From isolation to transformation, these methods are divided into three strategies. Lymphocytes are separated from fresh blood by density gradient centrifugation and EBV transformation directly6-8, named density gradient separation method in this study. However, other techniques overcome the difficulty of shipping fresh blood and use a procedure for freezing purified lymphocytes and subsequent thawing and EBV infection9-11. Cryopreserved whole blood can also be used for the isolation of B lymphocytes and transformation12,13. In addition to density gradient centrifugation to separate B lymphocytes, some researchers use a magnetic cell sorter to select B lymphocytes12. However, magnetic separation is an expensive, complex and time consuming process. Although these methods are useful to immortalize B lymphocytes with a high success rate, the need to treat a large number of clinical samples and the relatively small volume of blood require a simpler method for the establishment of a permanent cell line.
Another less commonly used method is using whole blood as the source of nucleated cells for EBV infection and the establishment of B-LCLs. These methods simplify the technique by omitting the separation procedure. Only a small amount of whole blood, either fresh14 or frozen15,16, is sufficient to obtain the cell lines. Unfortunately, there is great variability in success from documents15,16 and several of our validation tests. Furthermore, transformed colonies are hardly recognized under a microscope because of the large contaminating impurities. Thus, a more reliable method for transformation is required.
Based on the above considerations, we have developed a novel method to establish B-LCLs without previous purification of the lymphocytes, named the red blood cell lysis method. This method is convenient, time saving and applicable to a large number of samples. As little as 0.5 mL of whole blood was enough to obtain B-LCLs that are easily obtainable from infants and the elderly without significantly harming their health.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Techcomp</td>
			<td style="width:119px;">CT6T</td>
			<td style="width:167px;">Centrifugation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automated Cell Counter&#160;</td>
			<td style="width:71px;">Countstar</td>
			<td style="width:119px;">&#160;IC 1000&#160;</td>
			<td>For cell counting&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">&#160;Epoch Microplate Spectrophotometer</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;">SN263839</td>
			<td>For measuring the absorbance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">96 well cell culture cluster</td>
			<td style="width:71px;">Corning Incorporated</td>
			<td style="width:119px;">3599</td>
			<td>Polystyrene plates</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">24 well cell culture cluster</td>
			<td style="width:71px;">Corning Incorporated</td>
			<td style="width:119px;">3524</td>
			<td>Polystyrene plates</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">25cm2 cell culture flask</td>
			<td style="width:71px;">Nest</td>
			<td style="width:119px;">707001</td>
			<td>Polystyrene&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">10270</td>
			<td>Component of B-LCLs medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI Medium 1640</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31800-022</td>
			<td>For B-LCLs medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutamine&#160;&#160;</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0374</td>
			<td>Component of B-LCLs medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 x streptomycin penicillin solution BioRoYeeBRY-2309</td>
			<td style="width:71px;">BioRoYee</td>
			<td style="width:119px;">BRY-2309</td>
			<td>Component of B-LCLs medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ficoll paque plus</td>
			<td style="width:71px;">GE&#160; Healthcare</td>
			<td style="width:119px;">17-1440-03</td>
			<td>For in vitro isolation of lymphocyte</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PHA-M</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L8902</td>
			<td>For stimulating lymphocyte proliferation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cyclosporin A</td>
			<td style="width:71px;">Cayman&#160; Chemical</td>
			<td style="width:119px;">12088</td>
			<td>For inhibiting the cytotoxicity effect of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Red cell lysis buffer</td>
			<td style="width:71px;">Tiangen</td>
			<td style="width:119px;">RT122-01</td>
			<td>For lysing red blood cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0793</td>
			<td>For the detection of cell viability</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td>For freezing cells</td>
		</tr>
	</tbody>
</table>

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.

During the process of transformation, the morphological changes of cells are visualized by light microscopy (Figure 2). Small clusters of cells were clearly visible with the density gradient separation method after 7 days of infection, while only a thick layer of cell fragments are visible from the red blood cell lysis method. However, lymphoblastoid cell clusters are visible under the thick layer of cell fragments 30 days post infection. With more frequent medium change, the cell debris decreases and the cell clusters are clearly visible and present the same image established by the density gradient separation method.
When cell lines were established successfully, the MTT assay was used to evaluate the viability of B-LCLs. According to the literature17, B-LCLs established by different methods were selected to detect the cell viability and 1640 medium was used as blank control. The data indicate that the cell viability of B-LCLs established with the red blood cell lysis method is significantly higher than the cell viability of B-LCLs from the density gradient separation method (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/55191/55191fig1.jpg" /> 
Figure 1. Flow Chart of Procedure. Anticoagulant blood is treated with red blood cell lysis buffer before EBV infection followed by the addition of CsA. The infected B cells are grown at 37 &#176;C in the presence of 5% CO2 to establish and subsequently expand B-LCLs. <a href="http://cloudflare.jove.com/files/ftp_upload/55191/55191fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55191/55191fig2.jpg" /> 
Figure 2. Visualization of Cellular Morphological Changes in the Process of EBV Transformation. The left are B-LCLs established by red blood cell lysis method with 0.5 mL peripheral blood, and cell debris decreasing as time goes on. The right shows cell lines established by the density gradient separation method with 2.5 mL blood sample. Scale bar is 10 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55191/55191fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55191/55191fig3.jpg" /> 
Figure 3. Comparison of Cell Viability for B-LCLs Established by Different Methods. Each bar represents mean &#177; SD. The results show that there was significant difference between the two methods (p= 0.014). <a href="http://cloudflare.jove.com/files/ftp_upload/55191/55191fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.8K Views.  Chinese Academy of Sciences. 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.

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

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

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.

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 ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. 
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

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 ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...