Name: Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro (Video) | JoVE Journal
Uploaded: 2010-06-01T17:00:00.000+00:00
Duration: 8 min 39 s
Description: Learn to isolate and culture human T lymphocytes for in vitro migration analysis using ICAM-1 and SDF-1 substrates. This protocol enables researchers to study integrin-mediated immune cell dynamics.

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5 minutes

This project was supported by National Institutes of Health Grants HL087088 (M.K.) and HL18208 (M.K.).

1. Isolation of Human T Lymphocytes
<ol>
 <li>Obtain human blood from a healthy donor. Allow the blood to cool to room temperature (~30 min) before proceeding to the next step.</li>
 <li>Gently pipette 3 mL of room temperature Polymorph density gradient media into an 8 mL round-bottom polystyrene tube. Gently add 3 mL of whole blood on top of the Polymorph media. It is important to avoid mixing of the two reagents.</li>
 <li>Centrifuge the tubes at 500 x g for 45 minutes at room temperature.</li>
 <li>Following the centrifugation, the peripheral blood mononuclear cells (PBMC) have now separated from other blood components into the top cell layer. The PBMC layer appears, from the top down, as the first cloudy band.</li>
 <li>Carefully remove the clear yellow-colored upper phase of the blood, above the PBMC layer, and then use a P1000 micropipette to transfer the PBMC layer to a 15 mL or 50 mL conical tube.</li>
 <li>Wash the PBMC twice with PBS, centrifuging cells at 500 x g for 5 minutes each time. The supernatant will be somewhat cloudy after each wash.</li>
</ol>
2. Culture of Human T Lymphocytes
<ol>
 <li>Using a pipette, transfer the PBMC to a T-75 culture flask in 20 mL RPMI 1640 media containing 10% FBS, 1% penicillin/streptomycin, and 1 μg/mL phytohemagglutinin (PHA).</li>
 <li>Incubate at 37°C and 5% CO2 for at least 1 hour, and up to 24 hours. This step allows monocytes, which will be adherent to the flask surface, to be separated from the lymphocytes that remain in suspension. If a short incubation (1 hour) is used at this step, it is acceptable to use RPMI 1640 media containing 10% FBS and 1% penicillin/streptomycin without supplementing with PHA as specified in step 2.1.</li>
 <li>Carefully remove all of the media from the flask, add it to a 50 mL conical tube, and centrifuge at 500 x g for 5 minutes.</li>
 <li>Resuspend the cell pellet, which now primarily contains lymphocytes, and transfer the cells to a new T-75 flask containing 25 mL RPMI 1640 media containing 10% FBS, 1% penicillin/streptomycin, and 1 μg/mL PHA.</li>
 <li>Incubate at 37°C for 3 days (2 days if the initial incubation of PBMC was overnight). After 24 hours of growth, it may be necessary to add 15-20 mL of fresh media and transfer to a larger T-175 flask.</li>
 <li>After 3 days, use a pipette to remove the media and suspended lymphocytes from the flask and transfer to a 50 mL conical tube. Centrifuge at 500 x g for 5 minutes.</li>
 <li>Resuspend the cell pellet and transfer cells to a new T-75 or T-175 flask containing 25 mL (T-75) or 50 mL (T-175) RPMI 1640 with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL human IL-2 or IL-15.</li>
 <li>Grow lymphocytes for 4-7 days. If starting with a T-75 flask, the culture will need to be expanded and transferred to a T-175 flask after 1-2 days.</li>
</ol>
3. In Vitro Lymphocyte Migration Assay
<ol>
 <li>1 day before the migration assay, coat a glass-bottom 0.17 mm dish with 20 μg/mL Protein A or G in PBS overnight at 4°C.</li>
 <li>Wash the dish extensively with PBS.</li>
 <li>Immobilize human ICAM-1/Fc (10 μg/mL) and human SDF-1 (2 μg/mL) by adding these reagents in PBS solution to the dish and incubating 4 hours at room temperature.</li>
 <li>Prepare T lymphocytes by first determining the cell density in culture in the T-175 flask using a hemacytometer. Approximately 2-5 x 105 cells should be used per dish to achieve a cell density appropriate for migration analysis.</li>
 <li>Wash T lymphocytes twice with PBS and then resuspend in 1 mL L-15 media containing 1 mg/mL D-glucose.</li>
 <li>Wash the dish coated with ICAM-1/Fc and SDF-1 extensively with PBS.</li>
 <li>Transfer T lymphocytes in 1 mL media to the dish and maintain them at 37°C.</li>
</ol>
4. Capturing an Image Sequence Using NIS Elements Software
<ol>
 <li>Open NIS Elements software.</li>
 <li>In the camera settings menu, choose "2x2 binning" as the mode for both live imaging and image capture.</li>
 <li>Go to the Applications menu and choose Define/Run Experiment.</li>
 <li>Choose the length of time between images and the total length of the image capture sequence.</li>
 <li>Press the "Run" button to begin image acquisition.</li>
 <li>Cells can be tracked and migration parameters (velocity, path length, displacement, etc.) quantified using one of several software packages, including ImageJ, AutoQuant or Volocity (Figure 1).</li>
 <li>To generate a "spider web plot," the x-y coordinates for each cell and timepoint are collected (Figure 2) and projected onto a graph with a common starting point for each cell at the origin (Figure 3).</li>
</ol>
5. Representative Results
On day 6 of culture in the presence of either IL-2 or IL-15, T lymphocytes make up &gt;98% of cells, as determined by positive CD3 staining as well as positive CD4 and/or CD8 staining. For IL-2, we found 83% CD4+ cells, 15% CD8+ and &lt;1% CD4+CD8+ cells. For IL-15, we found 88% CD4+ cells, 11% CD8+ and &lt;1% CD4+CD8+ cells. During T lymphocyte migration on ICAM-1/SDF-1 substrates, cells exhibited a velocity of approximately 15 μm/min that can be sustained over a 1 hour time period. T lymphocyte migration on ICAM-1/SDF-1 is dependent on LFA-1-mediated adhesion, as an anti-LFA-1 ligand-blocking antibody severely inhibits migration.
<img src="/files/ftp_upload/2017/2017fig1.jpg" alt="Cell migration analysis; microscopy images showing cell paths with color-coded trajectories." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Cell tracking of migrating T lymphocytes. T lymphocytes isolated from whole blood and cultured in the presence of IL-2 or IL-15 for 6 days were allowed to adhere and migrate on glass-bottomed dishes coated with 10 μg/mL ICAM-1 and 2 μg/mL SDF-1. Images were acquired every 10 seconds for 30 minutes. Cells were identified in each image and tracked over time using Volocity software. This movie shows the random migration of T lymphocytes at a velocity of ~15 μm/min.
<a href="https://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">Click here to see the video.</a> 
Movie 1. Cell tracking of migrating T lymphocytes. T lymphocytes isolated from whole blood and cultured in the presence of IL-2 or IL-15 for 6 days were allowed to adhere and migrate on glass-bottomed dishes coated with 10 μg/mL ICAM-1 and 2 μg/mL SDF-1. Images were acquired every 10 seconds for 30 minutes. Cells were identified in each image and tracked over time using Volocity software. This movie shows the random migration of T lymphocytes at a velocity of ~15 μm/min.
<img src="/files/ftp_upload/2017/2017fig2.jpg" alt="Cell centroid tracking results; data table, position coordinates over time, cell migration analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Spatiotemporal cell position. The X-Y coordinates of each cell were obtained for each time point using Volocity software.
<img src="/files/ftp_upload/2017/2017fig3.jpg" alt="Random walk trajectories, scatter plot, statistical simulation, stochastic processes in data analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. "Spider web plot." Using the X-Y-t coordinates for each cell, 15 cells were chosen at random and plotted with a common starting point at the origin. Comparing spider web plots gives a quick visual depiction of differences in migration between different experimental conditions. Plots for T lymphocyte migration under control conditions (left panel) and in the presence of an anti-LFA-1 ligand-blocking antibody (right panel).

<ol><li>Hyun, Y. M., Chung, H. L., McGrath, J. L., Waugh, R. E., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activated+integrin+VLA-4+localizes+to+the+lamellipodia+and+mediates+t+cell+migration+on+VCAM-1%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Activated integrin VLA-4 localizes to the lamellipodia and mediates t cell migration on VCAM-1.</a> J Immunol. 183, 359-369 (2009).</li>
<li>Morin, N. A., Oakes, P. W., Hyun, Y. M., Lee, D., Chin, Y. E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonmuscle+myosin+heavy+chain+IIA+mediates+integrin+LFA-1+de-adhesion+during+T+lymphocyte+migration%5BTitle%5D)+AND+%22The+Journal+of+experimental+medicine%22%5BJournal%5D)">Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.</a> The Journal of experimental medicine. 205, 195-205 (2008).</li>
<li>Hyun, Y. M., Lefort, C. T., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukocyte+integrins+and+their+ligand+interactions%5BTitle%5D)+AND+%22Immunologic+research%22%5BJournal%5D)">Leukocyte integrins and their ligand interactions.</a> Immunologic research. , (2009).</li>
</ol>

No conflicts of interest declared.

In this experiment, we provide details on a simple system to analyze the motility of primary human T lymphocytes. In vitro migration assays have been used to dissect the roles of many molecules and signaling pathways involved in the locomotion of various cell types. Some critical controls to keep in mind when designing your own experiment from our protocol include: 1) Non-adhesive or non-integrin adhesive substrate coatings, such as bovine serum albumin (BSA) or poly-L lysine (PLL), respectively, 2) integrin-spe...

<table><tbody><tr><td>RPMI 1640</td><td>GIBCO, by Life Technologies</td><td>11875</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher Scientific, Inc.</td><td>SH300070.03</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>1-Step Polymorphs</td><td>Accurate Chemical &amp;amp; Scientific Corporation</td><td>AN221725</td><td></td></tr><tr><td>PHA</td><td>Remel, Thermo Fisher Scientific</td><td>R30852801</td><td></td></tr><tr><td>Rec. human IL-2</td><td>R&amp;amp;D Systems</td><td>202-IL</td><td></td></tr><tr><td>Rec. human IL-15</td><td>R&amp;amp;D Systems</td><td>247-IL</td><td></td></tr><tr><td>Protein G</td><td>Sigma-Aldrich</td><td>19459</td><td></td></tr><tr><td>Rec. human ICAM-1/Fc</td><td>R&amp;amp;D Systems</td><td>720-IC</td><td></td></tr><tr><td>Rec. human SDF-1</td><td>R&amp;amp;D Systems</td><td>350-NS</td><td></td></tr></tbody></table>

human t lymphocyte isolation, culture and analysis of migration in vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

T lymphocyte Migration occurs during homing to lymphoid organs, exit from the vasculature and entry into peripheral tissues. This process involves the adhesive interaction of the T-cell surface with other cells. To examine this phenomenon in vitro human T lymphocytes are isolated, cultured, and placed on tissue culture plates coated with the adhesive protein.ICAM one and chemokine SDF one. Images of T lymphocyte migration can then be acquired and analyzed. Hi, I'm Craig LeFort and the laboratory of Minsu Kim in the Center for Vaccine Biology and Immunology at the University of Rochester.Today we'll show you a procedure for the isolation and culture of human teen lymphocytes, an analysis of their migration in vitro. We use this assay in our laboratory to study the role of integrins and T-cell migration. The major integrin in T-cell is lymphocyte function associated antigen one or LFA one.The specific ligands for LFA one are the icas, such as ICAM one. In addition, a kin signal is required for T-cell migration to provide both a directionality signal as well as to activate integrins. In our assay, we use human ICAM one and CX CL 12 or SDF one as substrates for T-cell migration.So let's get started. After obtaining human blood from a healthy donor, allow the blood to cool to room temperature. This takes about 30 minutes.Once the blood is cooled, gently pipette three milliliters of room temperature, polymorph density gradient media into an eight milliliter round bottom polystyrene tube. Then gently add three milliliters of whole blood on top. It is important to avoid any mixing.Place the tubes in a centrifuge and spin it 500 G for 45 minutes. At room temperature following the centrifugation, the peripheral blood mononuclear cells or P BMCs have been separated from the other blood components. The PBMC layer appears as the first cloudy band from the top.Under sterile conditions, carefully remove and discard the clear yellow colored upper phase. Then use a P 1000 micro pipette to transfer the PBMC layer to a new conical tube. Wash the PBMC twice with PBS centrifuging cells at 500 G for five minutes.Each time the supernatant will be somewhat cloudy After each wash. Re suspend the cells in 20 milliliters of RPMI 1640 media containing 10%FBS, 1%penicillin streptomycin, and one microgram per milliliter. Phyto hemagglutinin or PHA incubation in the presence of PHA induces T lymphocyte activation and expansion.Using a pipette transfer the pbmc to a T 75 culture flask. Next incubated 37 degrees Celsius and 5%carbon dioxide for one to 24 hours. This step allows monocytes, which will adhere to the flask surface to be separated from the lymphocytes that remain in suspension.Following the incubation, carefully remove all of the media which contains primarily lymphocytes and transfer it to a 15 milliliter conical tube. Centrifuge at 500 G for five minutes. Reese has been the cell pellet in RPMI 1640 and transfer the cells to a new T 75 flaz containing 25 milliliters of RPMI 1640 with FBS, penicillin, streptomycin, and PHA incubated 37 degrees Celsius.After 24 hours of growth, it may be necessary to add 15 to 20 milliliters of fresh media and transfer to a larger T 1 75 flask. Continue incubating for three days or two days. If the initial incubation of PBMC was overnight after three days, use a pipette to remove the medium, which will contain suspended lymphocytes and transfer it to a 50 milliliter conical tube centrifuge at 500 G for five minutes.Reese has been the cell pellet and transferred the cells to a new T 75 containing 25 milliliters of RPMI 1640. With FBS, penicillin, streptomycin and human IL two or IL 15 place the cells in the incubator. If starting a T 75 flask, the culture will need to be expanded and transferred to a T 1 75 flask.After one to two days grow lymphocytes for a total of four to seven days. One day before the migration assay coat a glass bottom 0.17 millimeter dish with 20 micrograms per milliliter of protein A or G in PBS incubate overnight at four degrees Celsius the next day. Wash the dish extensively with PBS, then add ICAM one FC and human SD F1 in PBS solution to the dish.Incubate for four hours at room temperature to immobilize the molecules. Determine the density of the cultured cells using a hemo cytometer. Then wash the lymphocytes twice with PBS and resuspend them in one milliliter of L 15 media containing D glucose following the incubation, wash the coated dish with ICAM one FC and SDF one extensively with PBS transfer the T lymphocytes in one milliliter of media to the dish, approximately two to five times 10 to the fifth cell should be used per dish to achieve a cell density appropriate for migration analysis.To obtain images of cell migration, place the dish on the microscope stage in a temperature controlled environment such as a heated chamber at 37 degrees Celsius. Open the NIS elements software in the image settings menu. Choose two by two binning as the mode for both live imaging and image capture.Go to the applications menu and choose, define, run, experiment. Choose the length of time between images and the total time. For the image capture sequence, press the run button to begin image acquisition following the acquisition.Migration parameters such as velocity, path length and displacement can be quantified using a software package such as Image J Auto Quant or veloc. To generate a spider web plot, collect the XY coordinates for each cell and time point and project them onto a graph with a common starting point for each cell at the origin. The T lymphocytes shown here were cultured plated on ICAM and SDF one and imaged every 10 seconds for 30 minutes.This movie shows the random migration of T lymphocytes at a velocity of approximately 15 microns per minute using the XYT coordinates for each cell. 15 cells were chosen at random and plotted with a common starting point at the origin. Comparing spiderweb plots gives a quick visual depiction of differences in migration between different experimental conditions.Plots for T lymphocyte migration under control conditions are shown on the left while plots for T lymphocyte migration in the presence of an anti LFA one ligand blocking antibody are shown on the right. These data demonstrate that T lymphocytes utilize integrin LFA one to migrate on an ICAM one substrate. We've just shown you how to do a migration assay using cultured human T lymphocytes during the procedure.It's, it's important to remember to add an appropriate number of cells to the ICAM one coated dish so that cell tracking and analysis of migration are simplified. So that's it. Thanks for watching and good luck with your experiments.

human-t-lymphocyte-isolation-culture-analysis-migration

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Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

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