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>
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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 ...

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

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