Name: isolation and activation of murine lymphocytes
Uploaded: 2016-10-30T14:00:00.000+00:00
Duration: 8 min 8 s
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 Solution (BSS) Stock 1 and Stock 2, Separately.
	<ol>
		<li>Prepare 20x BSS stock 1 (111 mM dextrose, 8.8 mM potassium phosphate, 26.7 mM sodium phosphate dibasic in 1 L sterile water) and add 40 ml 0.5% Phenol Red to 20x BSS stock 1 before final volume adjustment. Sterile filter using 0.2 &#181;m filter before storing at 4 &#176;C.</li>
		<li>Prepare 20x BSS stock 2 (25.8 mM calcium chloride dihydrate, 107 mM potassium chloride, 2.73 M sodium chloride, 19.6 mM magnesium chloride hexahydrate, 16.6 mM magnesium sulfate in 1 L sterile water). Sterile filter using 0.2 &#181;m filter before storing at 4 &#176;C.</li>
		<li>To prepare 1x BSS for experimental use, dilute 50 ml BSS stock 1 and 50 ml BSS stock 2, separately, in 400 ml sterile water each. Combine both diluted solutions, adjust to pH 7, and add 20 ml FBS (2%). Top up to 1 L using sterile water and sterile filter using 0.2 &#181;m filter. 
		NOTE: BSS stock solutions should be prepared separately because the mixing of concentrated BSS stocks directly could result in precipitation.</li>
	</ol>
	</li>
	<li>Red Blood Cell (RBC) Lysis Buffer
	<ol>
		<li>To prepare RBC lysis buffer, mix 9 parts stock ammonium chloride (155 mM ammonium chloride in sterile water) to 1 part stock Tris-base (130 mM Tris(hydroxymethyl)aminomethane in sterile water, pH 7.65) before use. 
		NOTE: Store sterile stock solutions of ammonium chloride and Tris-base at 4 &#176;C. Prepare lysis buffer fresh to ensure efficient lysis of RBCs.</li>
	</ol>
	</li>
</ol>
2. Generation of Lymphocyte Suspension from Spleen or Lymph Nodes
NOTE: It is important to prepare all reagents and equipment required for the experiment before mouse euthanasia and to generate single cell suspensions of lymphocytes as soon as possible to maintain high cell viabilities.
<ol>
	<li>Euthanize experimental mouse by cervical dislocation or CO2 asphyxiation. 
	NOTE: From this step onwards, all experimental procedures should be performed aseptically.</li>
	<li>Dip the entire mouse into 70% ethanol before making any incisions. Remove the spleen and lymph nodes aseptically8, and place them in separate 15 ml tubes containing 5 ml ice-cold RPMI/FBS (RPMI with 2% FBS) or BSS/FBS (from step 1.2.3). 
	NOTE: Since BSS prevents efficient RBC lysis, use RPMI for the preparation of the splenic cell suspension and switch to BSS after RBC lysis.</li>
	<li>To generate a single cell suspension from spleen or lymph nodes, place the organ(s) in between two pieces of sterile 100 &#181;m cell strainer mesh in a petri dish containing 2 ml ice-cold RPMI/FBS or BSS/FBS. Using the plunger of a 1 ml syringe, mash the organ(s) until it has been torn into very fine parts.</li>
	<li>Transfer the cell suspension to a 15 ml tube and wash the cell strainer mesh with ice-cold RPMI/FBS or BSS/FBS. Collect the remaining cell suspension, add it to the same 15-ml tube, and spin down at 453 x g for 5 min at 4 &#176;C. Remove the supernatant. 
	NOTE: Re-suspend the pelleted cells by flicking the tube with fingers before adding RBC lysis buffer or medium in the subsequent steps.</li>
	<li>Prepare room temperature (RT) RBC lysis buffer during centrifugation of the cell suspension (see step 1.3.). After pelleting the cells and removing the supernatant, re-suspend the cells with 1 ml RBC lysis buffer for every 108 cells. Incubate the lysis reaction at RT for 3-4 min.</li>
	<li>Stop RBC lysis with 14 ml ice-cold BSS/FBS and spin down at 453 x g for 5 min at 4 &#176;C.</li>
</ol>
3. Purification of B and T Cells
<ol>
	<li>Purification of B Cells
	<ol>
		<li>Count the cells using a hemocytometer. Re-suspend up to 108 splenic cells in 300 &#181;l BSS/FBS and add 50 &#181;l anti-CD43 magnetic microbeads9,10. To remove dead cells, add 30 &#181;l Annexin V magnetic beads. Incubate the cell suspension in a 4 &#176;C refrigerator for 30 min.</li>
	</ol>
	</li>
	<li>Purification of T Cells
	<ol>
		<li>Count the cells using a hemocytometer. Re-suspend up to 108 cells in non-T cell depletion antibody cocktail (biotinylated antibodies against CD19, B220, Gr-1, TCR-&#947;&#948;, CD49b, CD11c, CD11b, Ter119 and CD4 or CD8 depending on the target cell population to be purified), diluted 1:200 in 200 &#181;l BSS/FBS4,5. Incubate the cell suspension in a 4 &#176;C refrigerator for 15 min.</li>
		<li>After incubation, add 10 ml BSS/FBS to wash the cells and spin down at 453 x g for 5 min at 4 &#176;C. Remove the supernatant and re-suspend the cells in 165 &#181;l BSS/FBS with 30 &#181;l streptavidin microbeads and 15 &#181;l Annexin V magnetic beads. Incubate the cell suspension in a 4 &#176;C refrigerator for 30 min. 
		NOTE: To ensure even labeling with the magnetic microbeads, incubate the cells with microbeads for 15 min, then mix the cell suspension gently by tapping the 15 ml tube and incubate another 15 min during step 3.1.1. or 3.2.2.</li>
	</ol>
	</li>
	<li>Preparation of Separation Column for Cell Purification
	<ol>
		<li>Prepare an unused separation column during microbead labeling of the cells (step 3.1.1. or 3.2.2.). Pre-warm BSS (without FBS) to RT and use 2 ml to wash and equilibrate the column aseptically. After equilibration with BSS, the washed column should not be allowed to dry out. 
		NOTE: We use the LS column instead of the recommended LD column due to its re-usability (see step 3.4.).</li>
		<li>After labeling with the magnetic beads, add 14 ml BSS/FBS to wash the cells and spin down at 453 x g for 5 min at 4 &#176;C. Remove the supernatant and re-suspend the cells in 1-3 ml RT BSS.</li>
		<li>Attach a sterile 21 G needle to the tip of the column to reduce the flow rate during the process of purification. Load the cell suspension onto the equilibrated column and collect the flow through containing the purified target cells. 
		NOTE: Avoid introducing bubbles into the column while loading.</li>
		<li>Wash the column once with 1 ml BSS/FBS and collect the flow through containing the purified target cells. Reload the column with the flow through once again. Collect the flow through after the second loading in the same 15 ml tube.</li>
		<li>Wash the column 3 times with 1 ml BSS/FBS and collect the flow through containing the purified target cells. Thereafter, add 5 ml BSS/FBS to the column and, with a plunger, flush the magnetically labeled cells out of the column into a new 15 ml tube.</li>
		<li>Check the purity of the cells collected by flow cytometry using antibodies that bind to surface antigens of purified B or T cells4,5.</li>
	</ol>
	</li>
	<li>Re-using the Separation Column 
	NOTE: The LS column can be reused up to 4 times without affecting purification efficiency.
	<ol>
		<li>Wash the column 3 times with 5 ml phosphate buffered saline (PBS) and 3 times with 5 ml distilled water from the top using the plunger.</li>
		<li>Wash the column with 5 ml 70% ethanol and dry the column extensively using an air tap to prevent the buildup of rust in the column.</li>
		<li>To prepare a used LS column for a separate purification experiment, wash the column from the bottom up with 5 ml 70% ethanol using the syringe adaptor. Next, wash the column from the bottom up twice with 5 ml sterile PBS, followed by 5 ml PBS once from the top of the column. Add 2 ml RT BSS to equilibrate the column and then proceed to loading the column with labeled cells.</li>
	</ol>
	</li>
</ol>
4. CFSE Labeling and Stimulation
NOTE: Purified cells can be subjected to a variety of in vitro and in vivo functional assays. Here, we use purified T cells to determine the T cell stimulation capability of APCs5.
<ol>
	<li>Pre-warm labeling solution (0.1% FBS in PBS) to 37 &#176;C prior to CFSE loading. 
	NOTE: Using a low percentage of FBS in PBS reduces cell death during CFSE loading and minimizes cell loss during centrifugation. However, too much FBS can interfere with CFSE loading.</li>
	<li>Wash purified cells twice with labeling solution, then re-suspended at 2 x 107 cells/ml in pre-warmed labeling solution in a 15 ml tube.</li>
	<li>Prepare 10 &#181;M CFSE solution (1:500 dilution of 5 mM CFSE stock solution) in pre-warmed labeling solution. CFSE solution should be freshly prepared each time to achieve optimal labeling.</li>
	<li>To load cells with CFSE, add 1 part cell suspension to 1 part 10 &#181;M CFSE solution in a 15-ml tube and incubate in the dark for 10 min at 37 &#176;C. A final concentration of 5 &#181;M CFSE is used to label 1 x 107 cells/ml.</li>
	<li>Invert the tube every 2 min to ensure a homogenous mixture of cells during CFSE loading.</li>
	<li>To stop the reaction, add several volumes of ice-cold complete RPMI medium and spin down at 453 x g for 5 min at 4 &#176;C. Upon successful CFSE loading, the cell pellet will appear yellowish.</li>
	<li>Wash CFSE loaded cells one more time with ice-cold complete RPMI medium and spin down at 453 x g for 5 min at 4 &#176;C before using for in vitro culturing or in vivo stimulation.</li>
</ol>
5. In Vitro Stimulation
<ol>
	<li>Prepare a 2x stock solution of stimuli (2x stimuli stock solution) immediately before use so that 100 &#181;l of 2x stimuli stock solution can be added to 100 &#181;l of cells to a final volume of 200 &#181;l per well in a 96-well plate.</li>
	<li>If a plate coated with stimuli (IgM or CD40 for B cells or CD3 and CD28 for T cells) is required, dilute the stimuli in PBS and pre-coat the culture plate at 4 &#176;C overnight. Alternatively, the culture plate can be coated at 37 &#176;C for 1 hr on the day of the experiment. Wash the coated plate twice with PBS (Do not allow the plate to dry out at any time).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>For B cells&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimuli&#160;</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>F(ab&#8217;)2 goat anti-mouse IgM</td>
			<td>0.6-2.4 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40 mAb</td>
			<td>0.5-2 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Recombinant mouse IL-4</td>
			<td>25 U/ml&#160;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide</td>
			<td rowspan="2">0.1-10 &#181;g/ml</td>
		</tr>
		<tr>
			<td>(LPS) from E. coli Serotype 055:B5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For T cells&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimuli&#160;</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Anti-CD3 (plate coated)</td>
			<td>2-10 &#181;g /ml</td>
		</tr>
		<tr>
			<td>(50 &#181;l/well for coating)</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD28 (plate coated)&#160;</td>
			<td>2 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Recombinant IL-2</td>
			<td>40 U/ml</td>
		</tr>
		<tr>
			<td>PDBu (Phorbol ester)</td>
			<td>5-50 ng/ml</td>
		</tr>
		<tr>
			<td>A23187 (Calcium ionophore)</td>
			<td>250 ng/ml</td>
		</tr>
	</tbody>
</table>
Table 1: Concentrations of stimuli used to stimulate lymphocytes in in vitro culture.
<ol start="3">
	<li>For CFSE-labeled B cells, re-suspend to 3 x 106 cells/ml in complete RPMI medium and culture in triplicate with 3 x 105 cells/well in 96-well flat bottom plates for 72 hr.</li>
	<li>For CFSE-labeled T cells, re-suspend to 0.5-3 x 106 cells/ml in complete RPMI medium and culture in triplicate with 0.5-3 x 105 cells/well in 96-well round bottom plates for 48 or 72 hr.</li>
</ol>
6. In Vivo Stimulation
<ol>
	<li>For in vivo stimulation, adoptively transfer 4 x 106 CFSE-labeled T cells per mouse (intravenously (i.v.) in 200 &#181;l PBS) into each MHC-matched recipient mouse. 
	NOTE: In this protocol, CFSE-labeled T cells can be adoptively transferred using tail vein or retro-orbital injection as these cells will home to lymphoid organs such as the spleen and lymph nodes.</li>
	<li>Challenge the recipient mice one day later with the antigen. 
	NOTE: In this example, we use ovalbumin (OVA protein, 50 &#181;g/mouse) as the antigen because OVA-specific, T cell receptor (TCR)-transgenic T cells were adoptively transferred into recipient mice. Prepare OVA protein in sterile PBS and inject 100 &#181;l of OVA protein/PBS or PBS control, via subcutaneous injection (s.i.), into each recipient mouse5.</li>
	<li>Harvest and generate single cell suspensions from lymphoid organs (lymph nodes and spleens) of recipient mice 3 days after immunization with OVA protein or PBS. Separate lymph nodes into proximal lymph nodes (pLN), which includes axillary, brachial and superficial cervical lymph nodes) and distal lymph nodes (dLN), which includes mesenteric, popliteal, inguinal, lumbar, and caudal lymph nodes. Stain cells using the appropriate FACS antibodies to check for T cell proliferation. 
	NOTE: In this CFSE cell tracking experiment, CFSE-labeled T cells from PBS-injected control mice establish a baseline fluorescence for non-dividing cells. Cell divisions of proliferating, antigen-stimulated, CFSE-labeled cells are visualized by measuring fluorescence peaks12,13. With the PBS control, the number of cell divisions of proliferating, CFSE-labeled T cells can be determined12,13.</li>
	<li>Analyze the CFSE-labeled cell proliferation data by comparing the number of cell divisions or peaks between samples (Figures 1 and 2). 
	NOTE: For example, CFSE-labeled, OVA-specific T cells adoptively transferred into recipient mice receiving the OVA antigen will undergo active proliferation compared to the PBS-injected control mice5,12. Furthermore, it is noteworthy to point out that there are many ways to analyze CFSE cell proliferation data, as demonstrated by Hawkins and colleagues14.</li>
</ol>

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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>

The authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></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&amp;nbsp;</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&amp;nbsp;</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 &amp;mu;m cell strainer mesh</td><td></td><td></td><td>To sterilize using UV radiation prior use</td></tr><tr><td>0.2 &amp;mu;m&amp;nbsp; 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></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&amp;nbsp;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies and recombinant protein&lt;/strong&gt;</td><td></td><td></td><td></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-&amp;gamma;&amp;delta; 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&amp;rsquo;)2 goat anti-mouse IgM (plate coated)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>115-006-075</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-mouse CD40 mAb (plate coated)</td><td>Pharmingen&amp;nbsp;</td><td>553722</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-4</td><td>ProSpec&amp;nbsp;</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&amp;nbsp;</td><td>16-0037-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-CD28 (clone clone 37.51) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0281-85</td><td>50 &amp;micro;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 ...

isolation-and-activation-of-murine-lymphocytes

Research

JoVE Journal

Immunology and Infection

21.0K Views.  Nanyang Technological University. 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.

Video: Isolation and Activation of Murine Lymphocytes

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 (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Cancer Research

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biology

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 bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

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

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

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

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Isolation and Activation of Murine Lymphocytes

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Rozdziały w tym wideo

Murine Model of CD40-activation of B cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Isolation of Murine Lymph Node Stromal Cells

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Murine Dermal Lymphatic Endothelial Cell Isolation