Name: isolation and activation of murine lymphocytes
Uploaded: 2016-10-30T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

The study is supported by the Ministry of Education, Singapore (AcRF Tier1-RG40/13 and Tier2-MOE2013-T2-2-038). The manuscript was edited by Amy Sullivan from Obrizus Communications.

All mice are bred and maintained under specific pathogen-free conditions and all mouse protocols are conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee.
1. Preparation of Buffers and Reagents
<ol>
	<li>Prepare complete Roswell Park Memorial Institute (RPMI) medium (10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 IU/ml)/streptomycin (100 &#181;g/ml), 55 &#181;M 2-mercaptoethanol).</li>
	<li>Prepare 20x Balanced Salt ...

<ol>
	<li>Nikolich-&#381;ugich, J., Slifka, M. K., Messaoudi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+important+facets+of+T-cell+repertoire+diversity.">The many important facets of T-cell repertoire diversity.</a> Nat. Rev. Immunol. 4 (2), 123-132 (2004).</li><li>LeBien, T. W., Tedder, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+lymphocytes:+how+they+develop+and+function.">B lymphocytes: how they develop and function.</a> Blood. 112 (5), 1570-1580 (2008).</li><li>Brownlie, R., Zamoyska, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+signaling+networks:+branched,+diversified+and+bound.">T cell receptor signaling networks: branched, diversified and bound.</a> Nat. Rev. Immunol. 13 (4), 257-269 (2013).</li><li>Neo, W. H., Lim, J. F., Grumont, R., Gerondakis, S., Su, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-rel+regulates+ezh2+expression+in+activated+lymphocytes+and+malignant+lymphoid+cells.">C-rel regulates ezh2 expression in activated lymphocytes and malignant lymphoid cells.</a> J. Biol. Chem. 289 (46), 31693-31707 (2014).</li><li>Gunawan, M., 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=The+methyltransferase+Ezh2+controls+cell+adhesion+and+migration+through+direct+methylation+of+the+extranuclear+regulatory+protein+talin.">The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.</a> Nat Immunol. 16 (5), 505-516 (2015).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171 (1), 131-137 (1994).</li><li>Cabatingan, M. S., Schmidt, M. R., Sen, R., Woodland, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na&#239;ve+B+lymphocytes+undergo+homeostatic+proliferation+in+response+to+B+cell+deficit.">Na&#239;ve B lymphocytes undergo homeostatic proliferation in response to B cell deficit.</a> J. Immunol. 169 (12), 6795-6805 (2002).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Th17+differentiation+of+na&#239;ve+CD4+lymphocytes.">Isolation and Th17 differentiation of na&#239;ve CD4 lymphocytes.</a> J. Vis. Exp. (79), e50765 (2013).</li><li>Su, I., 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=Ezh2+controls+B+cell+development+through+histone+h3+methylation+and+Igh+rearrangement.">Ezh2 controls B cell development through histone h3 methylation and Igh rearrangement.</a> Nat. Immunol. 4 (2), 124-131 (2003).</li><li>Mecklenbr&#228;uker, I., Saijo, K., Zheng, N., Leitges, M., Tarakhovsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C&#948;+controls+self-antigen-induced+B-cell+tolerance.">Protein kinase C&#948; controls self-antigen-induced B-cell tolerance.</a> Nature. 416 (6883), 860-865 (2002).</li><li>Rush, J. S., Hodgkin, P. D. <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+activated+via+CD40+and+IL-4+undergo+a+division+burst+but+require+continued+stimulation+to+maintain+division,+survival+and+differentiation.">B cells activated via CD40 and IL-4 undergo a division burst but require continued stimulation to maintain division, survival and differentiation.</a> Eur. J. Immunol. 31 (4), 1150-1159 (2001).</li><li>Quah, B. J. C., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat. Protoc. 2 (9), 2049-2056 (2007).</li><li>Quah, B. J. C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> J. Immunol. Methods. 379 (1-2), 1-14 (2012).</li><li>Hawkins, E. D., Hommel, M., Turner, M. L., Battye, F. L., Markham, J. F., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+lymphocyte+proliferation,+survival+and+differentiation+using+CFSE+time-series+data.">Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.</a> Nat. Protoc. 2 (9), 2057-2067 (2007).</li><li>Tomlinson, M. J., Tomlinson, S., Yang, X. B., Kirkham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation:+Terminology+and+practical+considerations.">Cell separation: Terminology and practical considerations.</a> J. Tissue Eng. 4 (1), 1-14 (2013).</li></ol>

In this protocol, we demonstrate a procedure for purifying lymphocytes from lymphoid organs. Cell purification using magnetic bead sorting is a fast and simple method that yields viable, highly purified target cells.
Critical Steps within the Protocol
Cell viability and cell yield
Maintaining viability of hematopoietic lineage cells in vitro is critical to ensuring successful and reproducible experiments. Chemical ...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
		</tr>
		<tr>
			<td>RPMI 1640 (without L-Glutamine)</td>
			<td>Gibco</td>
			<td>31870025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td></td>
			<td></td>
			<td>Heat inactivated&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140114</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD43 magnetic microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-801</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Streptavidin microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Anti-Annexin V magnetic beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-201</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>MACS LD&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well U-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>650180</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well F-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer mesh</td>
			<td></td>
			<td></td>
			<td>To sterilize using UV radiation prior use</td>
		</tr>
		<tr>
			<td>0.2 &#956;m&#160; sterile disposable filter units</td>
			<td>Nalgene</td>
			<td>567-0020</td>
			<td>Can be substituted with any sterile filter device</td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>Invitrogen</td>
			<td>C34557</td>
			<td>CTV for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Yellow</td>
			<td>Invitrogen</td>
			<td>C34567</td>
			<td>CTY for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Far Red</td>
			<td>Invitrogen</td>
			<td>C34564</td>
			<td>CTFR for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>Cell Proliferation Dye eFluor 670</td>
			<td>eBioscience</td>
			<td>65-0840</td>
			<td>CPD670 for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>PKH26</td>
			<td>Sigma Aldrich</td>
			<td>PKH26GL</td>
			<td>PKH26, alternative to CFSE</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck Millipore</td>
			<td>S7653</td>
			<td>Can use from other sources</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>Molecular Probes</td>
			<td>C-1157</td>
			<td>Reconstitute in DMSO</td>
		</tr>
		<tr>
			<td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td>
			<td>Sigma Aldrich</td>
			<td>P1269</td>
			<td></td>
		</tr>
		<tr>
			<td>A23187 (Calcium ionophore)</td>
			<td>Sigma Aldrich</td>
			<td>C7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies and recombinant protein</td>
		</tr>
		<tr>
			<td>CD11b biotin (clone m1/70)</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD11c biotin (clone N418)</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Gr-1 biotin (clone RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Ter119 biotin (clone Ter119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>TCR-&#947;&#948; biotin (clone GL-3)</td>
			<td>Biolegend</td>
			<td>118103</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD19 biotin (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>B220 biotin (clone RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD49b biotin (clone DX5)</td>
			<td>Biolegend</td>
			<td>108904</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD4 biotin (clone GK1.5)</td>
			<td>Biolegend</td>
			<td>100404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD8 biotin (clone 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>F(ab&#8217;)2 goat anti-mouse IgM (plate coated)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>115-006-075</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40 mAb (plate coated)</td>
			<td>Pharmingen&#160;</td>
			<td>553722</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-4</td>
			<td>ProSpec&#160;</td>
			<td>Cyt-282</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from E. coli Serotype 055:B5</td>
			<td>Sigma Aldrich</td>
			<td>L-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD3 (clone clone OKT3) (plate coated)</td>
			<td>eBioscience&#160;</td>
			<td>16-0037-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-CD28 (clone clone 37.51 ) (plate coated)&#160;</td>
			<td>eBioscience&#160;</td>
			<td>16-0281-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-2</td>
			<td>ProSpec</td>
			<td>Cyt-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Ovalbumin</td>
			<td>Sigma Aldrich</td>
			<td>A7641</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and activation of murine lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Magnetic cell purification of lymphocytes allows users to purify a target cell population in a relatively short amount of time. Using our depletion protocol, we were able to increase the percentage of CD8 T cells (OT-I in recombination-activating gene-1 (RAG-1)-deficient mice) from 72.8% (before purification) to 94.2% (after purification; Figure 1A)4,5. These purified lymphocytes can then be used for downstream functional assays to determine lymphocyte prolifer...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

The overall goal of this experiment is to study the functional capacities of purified murine lymphocytes using various in vitro ad in-vivo functional assays. This method can help answer key questions in the fields of Immunology and Molecular Biology. Such as investigating function of a protein of interest in gene deficient lymphocytes.The main advantage of this technique is the ability to quickly purify lymphocytes with minimal manipulation and environmental stress. To purify B cells re-suspend up to one times ten to the eighth red blood cell liced whole spleen cells in 300 microliters of balanced salt solution or BSS supplemented with FBS. Then add 50 microliters of anti-CD43 magnetic micro beads.To remove the dead cells, add 30 microliters of Annexin five magnetic beads for 30 minutes at four degrees celsius. Flick the cell suspension at 15 minute intervals to ensure even labeling of cells. At the end of the incubation was the B labeled cells with 14 milliliters of BSS supplemented with FBS.After centrifugation, remove the supernatant, flick the tube and re-suspend the pellet in one to three milliliters of room temperature BSS supplemented with FBS. During the centrifugation load a separation column onto a magnetic rack and attach a sterile 21 gauge needle to the tip of the column to reduce the flow rate. Equilibrate the column with two milliliters of room temperature BSS.After re-suspending the pellet in one to three milliliters of room temperature BSS place the collection tube under the needle then load the B labeled cells onto the equilibrated column. Collect the flow through in a 15 milliliter conical tube. Then rinse the column with one milliliter of fresh BSS supplemented with FBS.Pooling the wash with the collected target cells reload the column with the target cell containing aluet, collecting the flow through in the same 15 milliter tube. And wash the column three times with one milliliter of BSS supplemented with FBS, continuing to pool the washes with a target cell suspension. After the third wash, remove the column from the magnet and load it with five milliliters of BSS supplemented with FBS.Using the column plunger to flush the magnetically labeled non target cells into a new 15 milliliter tube. Then, check the purity of the separated cells by flow cytometry. To re-use a separation column, after flushing the magnetic bead labeled cells wash the column three times from the top with five milliliters of PBS per wash and three times with five milliliters of distilled water per wash.Next, wash the column with five milliliters of 70 percent ethanol in the same way and use an air tap to thoroughly dry the column before storage. To re-use the column in a new purification experiment re-wash the column from the bottom up with five milliliters of 70 percent ethanol followed by two five milliliter PBS washes. Then load the top of the column with five milliliters of PBS for one final wash.And equilibrate the column with two milliliters of room temperature BSS. The column can then be loaded with the experimental cell population. To label the B purified cells with CFSE wash the cells two times with freshly prepared labeling solution in a 15 milliliter conical tube.Re-suspending the second pellet at two times 10 to the seventh cells per milliliter concentration in fresh 37 degrees celsius labeling solution. Each time, centrifuge the samples remove the supernatant and flick the tubes. Then label the cells at a one part cell suspension to one part freshly prepared 10 micromolar CFSE solution ratio in the dark for 10 minutes at 37 degrees celsius.Inverting the tube every two minutes to ensure an even distribution of the dye. Caution should be taken to ensure that the cell pellet is thoroughly suspended in a labeling solution. Cell clumps can affect the homogeneity of the CFSE labeling.At the end of the incubation add RPMI medium to quench CFSE and wash the cells two times in several volumes of ice cold complete RPMI medium. If the cells have been successfully labeled the pellet will be yellow in color. Next, wash a 96 well flat bottom plate coated with the appropriate stimulus two times in PBS.For CFSE labeled B cells, re-suspend to three times 10 the sixth B cells per milliliter and complete RPMI medium and seed 100 microliters of B cells per well onto the coated plate in triplicate for 72 hours. By this depletion method the purity of the bead isolated OT1-CD8 T cells can be increased from 72.8 percent to 94.2 percent. The cells can then be labeled with CFSE as just demonstrated and adoptively transferred into recipient animals for analysis of their in-vivo proliferation.In addition, anti-CD45.1 antibody labeling can be used to further confirm the identity of the CFSE expressing cells as the adoptively transferred donor CD-45.1 positive OT1-CD8 T cells in the lymphoid organs of recipient mice. Alternatively, the purified T cells can be stimulated in vitro and their proliferation can be assessed by tritiated Thymidine incorporation. A significant increase in splenic B cell purity is also achieved by this method.Facilitating similar opportunities for downstream functional analysis of the purified B cells. Once mastered, this technique can be completed in two hours. By performing this procedure, it is important to keep your CI suspension on ice at all times except during purification and CFSE labeling.Purified lymphocytes can also be used in other functional assays such as immunoblotting to the terminus signaling capacity of genetically modified lymphocytes. After watching this video you should have a better idea of how to purify B and T cells and how to use the cells for various in vitro and in vivo functional assays.

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...