The authors thank Dr. Julia Mannheim, Walter Ehrlichmann, Ramona Stumm, Funda Cay, Daniel Bukala, Maren Harant as well as Natalie Altmeyer for the support during the experiments and data analysis. This work was supported by the Werner Siemens-Foundation, the DFG through the SFB685 (project B6) and Fort&#252;ne (2309-0-0).

Safety Precautions: When handling radioactivity, store 64Cu behind 2-inch-thick lead bricks and use respective shielding for all vessels carrying activity. Use appropriate tools to indirectly handle unshielded sources to avoid direct hand contact and minimize exposure to radioactive material. Always wear radiation dosimetry monitoring badges and personal protection equipment and check oneself and the working area for contamination to immediately address it. Discard potentially contaminated personal protection ...

<ol>
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This protocol presents a reliable and easy method to stably radiolabel cells for in vivo tracking by PET. Utilizing this method, cOVA-TH1 cells, isolated and expanded in vitro from donor mice, could be radiolabeled with 64Cu-DOTA-KJ1-26-mAb and their homing was tracked to the pulmonary and perithymic LNs as sites of cOVA presentation in a cOVA-induced acute airway DTHR.
The modification of the mAb with the chelator requires fast and efficient working and the use of...

Non-invasive cell tracking is a versatile tool to monitor cell function, migration and homing in vivo. Recent cell tracking studies have focused on mesenchymal1,2 or bone-marrow derived stem cells3 in the context of regenerative medicine, autologous peripheral white blood cells in inflammation or T lymphocytes in adoptive cell therapies against cancer3,4. The elucidation of the sites of action and the underlying biological principles of cell-based therapies is of tremendous importance. CD8+ cytotoxic T lymphocytes, genetically engineered chimeric antigen receptor (CAR) T cells or tumor-infiltrating lymphocytes (TILs) were widely considered as the gold standard. However, tumor-associated antigen-specific TH1 cells have proven to be an effective alternative treatment option4,5,6,7.
As key players in inflammation, organ-specific autoimmune diseases (e.g., rheumatoid arthritis or bronchial asthma), and cells of high interest in cancer immunotherapy, it is important to characterize the temporal distribution and homing patterns of TH1 cells. Noninvasive in vivo imaging by PET presents a quantitative, highly sensitive method8 to examine cell migration patterns, in vivo homing, and the sites of T cell action and responses during inflammation, allergies, infections or tumor rejection9,10,11.
Clinically, 111In-oxine is used for leukocyte scintigraphy for the discrimination of inflammation and infection12, while 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is commonly used for cell tracking studies by PET3,13. One major disadvantage of this PET tracer, however, is the short half-life of the radionuclide 18F at 109.7 min and the low intracellular stability that impedes imaging at later time points post adoptive cell transfer. For longer term in vivo cell tracking studies by PET, although unstable in the cells, 64Cu-PTSM is frequently used to nonspecifically label cells14,15 with minimized detrimental effects on T cell viability and function16.
This protocol describes a method to further reduce disadvantageous effects on cell viability and function using a T cell receptor (TCR)-specific radiolabeled mAb. First, the production of the radioisotope 64Cu, the conjugation of the mAb KJ1-26 with the chelator DOTA, and the subsequent 64Cu-radiolabeling are shown. In a second step, the isolation and expansion of cOVA-TH1 cells of DO11.10 donor mice and the radiolabeling with 64Cu-loaded DOTA-conjugated mAb KJ1-26 (64Cu-DOTA-KJ1-26) are described in detail. The assessment of uptake values and efflux of radioactivity with a dose calibrator and by &#947;-counting, respectively, as well as the evaluation of the effects of 64Cu-radiolabeling on cell viability by trypan blue exclusion and functionality with IFN-&#947; ELISA are presented. For non-invasive in vivo cell tracking, the elicitation of a mouse model of cOVA-induced acute airway DTHR and image acquisition by PET/CT after adoptive cell transfer are described.
Moreover, this labeling approach can be transferred to different disease models, murine T cells with different TCRs or general cells of interest with membrane-bound receptors or expression markers underlying continuous membrane shuttling17.

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">HCl, Suprapur</td>
			<td style="width:227px;">Merck, Darmstadt, Germany</td>
			<td style="width:259px;">1.00318</td>
			<td style="width:189px;">64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Methanol, Suprapur</td>
			<td style="width:227px;">Merck, Darmstadt, Germany</td>
			<td>1.06007</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Isopropanol, Suprapur</td>
			<td style="width:227px;">Merck, Darmstadt, Germany</td>
			<td>1.0104</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Pt/Ir (90/10) plate</td>
			<td style="width:227px;">&#214;gussa</td>
			<td style="width:259px;">Custom made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">PEEK chamber</td>
			<td style="width:227px;">&#214;gussa</td>
			<td style="width:259px;">Custom made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">64Ni</td>
			<td style="width:227px;">Chemotrade</td>
			<td style="width:259px;"></td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Polygram SIL G/UV 254 plate</td>
			<td style="width:227px;">Macherey-Nagel</td>
			<td style="width:259px;">805021</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Ion exchange column</td>
			<td style="width:227px;">BioRad</td>
			<td style="width:259px;">AG1-X8</td>
			<td>64Cu production</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Solid state target system for PETtrace</td>
			<td style="width:227px;">WKL</td>
			<td style="width:259px;">costum made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">64Cu work-up module</td>
			<td style="width:227px;">WKL</td>
			<td style="width:259px;">costum made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Dose calibrator</td>
			<td style="width:227px;">Capintec</td>
			<td style="width:259px;">CRC-25R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">PETtrace cyclotron</td>
			<td style="width:227px;">General Electric Medical Systems</td>
			<td style="width:259px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DOTA-NHS</td>
			<td style="width:227px;">Macrocyclics</td>
			<td style="width:259px;">B-280</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Anti-cOVA-TCR antibody (KJ1-26)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">Na2HPO4</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">71633</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">H+ Chelex 100</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">C7901</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Amicon Ultra-15 filter unit</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">UFC910008</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Rotipuran ultrapure water</td>
			<td style="width:227px;">Carl Roth</td>
			<td style="width:259px;">HN68.3</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Ammonium acetate</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td>32301</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">PBS</td>
			<td style="width:227px;">University Tuebingen</td>
			<td style="width:259px;"></td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Micro Bio-spin P-6 column</td>
			<td style="width:227px;">Bio-Rad Laboratories</td>
			<td style="width:259px;">7326221</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Sodium citrate</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">71497</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Cyclone Plus PhosphorImager&#160;</td>
			<td style="width:227px;">Perkin-Elmer</td>
			<td>L2250116</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DMEM</td>
			<td style="width:227px;">Merck Millipore</td>
			<td>102568</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">FCS</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">S0115/1004B</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Sodium pyruvate</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">L0473</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">MEM-amino acids</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">K0293</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">HEPES&#160;</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">L 1613</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">&#160;Penicillin/Streptomycin</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">A2212</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">0.05 mM 2-&#946;-mercaptoethanol</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">M3148</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DO11.10 mice</td>
			<td style="width:227px;"></td>
			<td>in-house breeding</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DPBS</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:259px;">14190144</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Cell strainer 40 &#181;m&#160;</td>
			<td style="width:227px;">Corning</td>
			<td style="width:259px;">352340</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">ACK Lysing Buffer</td>
			<td style="width:227px;">Lonza</td>
			<td style="width:259px;">10-548E</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">CD4 MicroBeads, mouse</td>
			<td style="width:227px;">Miltenyi Biotech</td>
			<td style="width:259px;">130-097-145</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">QuadroMACS separator</td>
			<td style="width:227px;">Miltenyi Biotech</td>
			<td style="width:259px;">130-090-976</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">LS column</td>
			<td style="width:227px;">Miltenyi Biotech</td>
			<td style="width:259px;">130-042-401</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">anti-CD4 antibody (Gk1.5)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">anti-CD8 antibody (5367.2)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Anti-rat antibody (MAR18.5)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Rabbit complement MA</td>
			<td style="width:227px;">tebu-Bio</td>
			<td style="width:259px;">CL3221</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Anti-IL-4 antibody (11B11)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">cOVA 323-339-peptide&#160;</td>
			<td style="width:227px;">EMC-micro-collections</td>
			<td>Custom order</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">CPG1668-oligonucleotides</td>
			<td style="width:227px;">Eurofins MWG Operon</td>
			<td>Custom order</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">IL-2</td>
			<td style="width:227px;">Novartis</td>
			<td>65483-116-07</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">96-well plates</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>655180</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">24-well plates</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>662160</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">cell culture flask</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>660175</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">48-well plates</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>677 180</td>
			<td>cell labeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Gammacell 1000</td>
			<td style="width:227px;">Best Theratronics</td>
			<td>via inquiry&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gulmay RT225</td>
			<td style="width:227px;">Gulmay</td>
			<td>via inquiry&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Trypan blue</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">L6323</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Mouse IFN-&#947; ELISA</td>
			<td style="width:227px;">BD Biosciences</td>
			<td style="width:259px;">558258</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">PE Annexin V Apoptosis Detection Kit&#160;</td>
			<td style="width:227px;">BD Biosciences</td>
			<td style="width:259px;">559763</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Tube 5 ml</td>
			<td style="width:227px;">Sarstedt</td>
			<td style="width:259px;">55.476</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Round-bottom tubes&#160;</td>
			<td style="width:227px;">BD Biosciences</td>
			<td style="width:259px;">352008</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Wizard &#947;-counter</td>
			<td style="width:227px;">Perkin-Elmer</td>
			<td>2480-0010</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">ELISA Reader MultiscanEX</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td>51118177</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Microscope</td>
			<td style="width:227px;">Leica</td>
			<td>via inquiry&#160;</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">BD LSRII&#160;</td>
			<td style="width:227px;">BD Biosciences</td>
			<td>via inquiry&#160;</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">BALB/c mice</td>
			<td style="width:227px;">Charles River</td>
			<td style="width:259px;">028</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Aluminum gel</td>
			<td style="width:227px;">Serva Electrophoresis</td>
			<td style="width:259px;">12261.01</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Xylazine</td>
			<td style="width:227px;">Bayer HealthCare</td>
			<td>Ordered via University hospital</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Ketamine</td>
			<td style="width:227px;">Ratiopharm</td>
			<td>Ordered via University hospital</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Isoflurane</td>
			<td style="width:227px;">CP-Pharma</td>
			<td>Ordered via University hospital</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">30G needle</td>
			<td style="width:227px;">BD Biosciences</td>
			<td>304000</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Syringe</td>
			<td style="width:227px;">BD Biosciences</td>
			<td>11612491</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Capillaries 10 &#181;l</td>
			<td style="width:227px;">VWR</td>
			<td>612-2439</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Inveon PET scanner</td>
			<td style="width:227px;">Siemens Healthineers</td>
			<td style="width:259px;">no longer available</td>
			<td>in vivo cell trafficking, alternative companies: Bruker, Mediso&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Inveon SPECT/CT scanner</td>
			<td style="width:227px;">Siemens Healthineers</td>
			<td style="width:259px;">no longer available</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Inveon Research Workplace</td>
			<td style="width:227px;">Siemens Healthineers</td>
			<td style="width:259px;"></td>
			<td>image analysis, alternative software: Pmod</td>
		</tr>
	</tbody>
</table>

murine lymphocyte labeling by 64cu-antibody receptor targeting for in vivo cell trafficking by pet/ct

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Figure 1 summarizes the labeling of cOVA-TH1 cells with the 64Cu-DOTA-KJ1-26-mAb and the experimental design for the in vitro and in vivo studies covered in this protocol.
<img alt="Figure 1" src="/files/ftp_upload/55270/55270fig1.jpg" /> 
Figure 1: 64Cu-DOTA-KJ1-26-mAb Labeling Process &#38; Experimental Design. (A) Schematic represe...

Watch this Scientific Journal Video about Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT at JoVE.com

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

The overall goal of this procedure is to stably label murine lymphocytes with a copper-modified radioactive monoclonal antibody to track lymphocyte temporal distribution and homing patterns in vivo by PET/CT in mouse models of inflammation or cancer. This method can help elucidate the sites of action and the key underlying biological principles of cell-based therapies, such as stem cell therapies in regenerative medicine or cancer immunotherapies. The main advantage of this technique is that it permits the imaging of cell migration over 48 hours with a high contrast and minimal detrimental effects on the cells.This method can be easily transferred to any cell type of interest with specific membrane-bound receptors and corresponding monoclonal antibodies. Generally, individuals new to this method may struggle with the chelator conjugation and radiolabeling processes, resulting in a lower immunoreactive fraction and cell-specific activity. Begin by disinfecting a DO11.10 mouse with 70%ethanol.Then fix the limbs with adhesive tape, and make a medial incision along the abdomen of the animal. Laterally pull both sides of the incision, separate the skin from the peritoneum, and locate the cervical, axial, brachial, and inguinal lymph nodes. Using blunt forceps, transfer the lymph nodes into a container of 1%FCS in PBS.Then open the peritoneum, separate the spleen from the pancreas and connective tissue, and store the spleen with the lymph nodes. When all of the tissues have been collected, transfer the samples onto a 40-micron filter in a 15-milliliter conical tube, and use a syringe plunger to macerate the tissues. Then rinse the filter with 10 milliliters of FCS in PBS, and collect the single-cell suspension by centrifugation.After lysing the red blood cells, resuspend the white blood cell pellet in fresh PBS plus FCS and CD4-positive MicroBeads according to the manufacturer's instructions. After 20 minutes at four degrees Celsius, wash the cells in up to 50 milliliters of fresh PBS plus FCS buffer, and resuspend the cells in the appropriate volume of PBS plus FCS for magnetic column elution of the CD4-negative cell populations. Load the cells onto a magnetic cell separation column, and collect the flow-through in a 15-milliliter conical tube.At the end of the magnetic cell separation, use the plunger to flush the CD4-positive cell population into a new conical tube, and adjust the isolated CD4-positive T cell suspension concentration to one times 10 to the six cells per milliliter in fresh complete medium for four degrees Celsius storage. Collect the CD4-negative cells by centrifugation. Resuspend the pellet in anti-CD4, anti-CD8, mouse anti-rat monoclonal antibodies, and rabbit complement, and incubate for 45 minutes at 37 degrees Celsius.After collecting the cells by centrifugation, resuspend the pellet in three milliliters of fresh complete medium, and irradiate the negatively selected antigen-presenting cells at 30 grays. Adjust the antigen-presenting cell concentration to five times 10 to the six cells per milliliter in fresh complete medium, and add 100 microliters of CD4-positive T cells and 100 microliters of antigen-presenting cells into the appropriate experimental wells of a 96-well flat bottom plate. Then add the appropriate stimuli to each well, and transfer the plate to a cell culture incubator.For chicken ovalbumin-specific TH1 cell radiolabeling, first use a dose calibrator to draw 37 megabecquerels of the T cell-specific radioactive antibody of interest into a syringe without dead volume. Next, dispense the antibody into a reaction cup, and measure and subtract the remaining amount of reactivity in the syringe from the amount that was drawn. Then add one milliliter of saline to the cup to generate a 37-megabecquerel-per-milliliter solution.Next, add two times 10 to the six OVA-TH1 cells in 0.5 milliliters of complete medium to each well of a 48-well plate, followed by 20 microliters of the radiolabeled antibody solution. After 30 minutes in a radiation-safe, 37-degrees Celsius, and 7.5%carbon dioxide cell culture incubator, pool the radiolabeled cells in a 50-milliliter conical tube for two washes in 10 milliliters of 37 degrees Celsius PBS. To measure the radiolabeled antibody uptake and efflux, immediately after the second wash, transfer one times 10 to the six monoclonal antibody-labeled OVA-TH1 cells in one milliliter of complete medium into each of 10 gamma counting tubes.Pellet the cells by centrifugation, and transfer the supernatants into 10 new gamma counting tubes. Next, wash the cells in one milliliter of complete medium to remove any unbound radiolabeled monoclonal antibody, and transfer the supernatants into 10 new gamma counting tubes. Then add one milliliter of fresh complete medium to the cells, and measure the initial uptake values in a gamma counter.For in vivo PET/CT imaging, immediately after radiolabeling, adjust the OVA-TH1 cells to a five times 10 to the seven cells per milliliter concentration in PBS, and draw 200 microliters of cells into a one-milliliter syringe equipped with a 30-gauge needle. Inject the cells intraperitoneally into an ova delayed-type hypersensitivity reaction diseased animal between the fourth and fifth nipple. Then, to facilitate co-registration of the PET and CT images during the image analysis, fix glass capillaries containing radiolabeled antibody solution under a small animal bed.When the appropriate level of sedation is reached, apply ointment to the animal's eyes and use cotton swabs and surgical tape to immobilize the mouse on the bed. Mouse the mouse bed to the PET scanner, and center the field of view with a focus on the lungs to acquire a 20-minute static PET scan with an energy window of 350 to 650 kiloelectron volts. Transfer the mouse bed to the CT scanner.Via scout view, center the field of view on the lungs. Acquire a planar CT image via 360 projections during a 360-degree rotation in the step-and-shoot mode with an exposition time of 350 milliseconds and a binning factor of four. The applied radioactive dose of 0.7 megabecquerels reduces the TH1 cell viability by only 8%while higher doses demonstrate an even more pronounced effect.Compared to radiolabeling with copper-PTSM however, no significant advantage for the radiolabeled antibody is observed. T cell-specific radioactive antibody-labeled TH1 cells demonstrate no loss in functionality as evidences by interferon gamma secretion, a decreased efflux of radioactivity, and a reduced induction of apoptosis compared to copper-PTSM-labeled TH1 cells. Here, representative high-resolution static PET and anatomical CT images after adoptive transfer of the monoclonal antibody radiolabeled cells are shown, with the images fused in the coronal, axial, and sagittal view with the help of glass capillaries placed as just demonstrated.Volumes of interest were drawn on the pulmonary and perithymic lymph nodes on the decay-corrected and normalized PET images to calculate the percentage of injected dose per cubic centimeter. OVA-TH1 cell migration tracking to the pulmonary and perithymic lymph nodes as ova presentation sites in an airway delayed-type hypersensitivity reaction reveals that the in vivo PET signals derived from the monoclonal antibody radiolabeled OVA-TH1 cells are considerably higher than the signals from the copper-PTSM-labeled cells. Further, the OVA-TH1 cell uptake values were significantly increased in the delayed-type hypersensitivity reaction diseased animals compared to their untreated control littermates.Once mastered, this technique can be used to study the specific roles and sites of action of immune cells in animal models of human disease. While attempting this procedure, it's important to remember that each cell type might react differently to radioactivity and to adjust the amount of radioactivity used for the labeling accordingly. After watching this video, you should have a good understanding of how to isolate and culture antigen-specific T cells, label these cells with a radioactive antibody, and image their distribution and homing kinetics with PET/CT.Don't forget that working with radioactivity can be extremely hazardous and that precautions such as using proper lead shielding, minimizing exposure times, and maximizing the distance to the radioactive source should always be taken while performing this procedure.

murine-lymphocyte-labeling-64cu-antibody-receptor-targeting-for-vivo

Research

JoVE Journal

Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...