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.
1. Isolation of Human T Lymphocytes
2. Culture of Human T Lymphocytes
3. In Vitro Lymphocyte Migration Assay
4. Capturing an Image Sequence Using NIS Elements Software
5. Representative Results
On day 6 of culture in the presence of either IL-2 or IL-15, T lymphocytes make up >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 <1% CD4+CD8+ cells. For IL-15, we found 88% CD4+ cells, 11% CD8+ and <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.
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.
Click here to see the video.
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.
Figure 2. Spatiotemporal cell position. The X-Y coordinates of each cell were obtained for each time point using Volocity software.
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).
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-specific blocking antibody treatment to determine integrin specificity, and 3) co-coating without stimulatory signal, such as SDF-1 described in our protocol.
The authors have nothing to disclose.
This project was supported by National Institutes of Health Grants HL087088 (M.K.) and HL18208 (M.K.).
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
RPMI 1640 | Gibco | 11875 | ||
FBS | Thermo Scientific | SH300070.03 | ||
Penicillin-Streptomycin | Gibco | 15140 | ||
1-Step Polymorphs | Accurate Chemical | AN221725 | ||
PHA | Remel/Fisher Scientific | R30852801 | ||
Rec. human IL-2 | R&D Systems | 202-IL | ||
Rec. human IL-15 | R&D Systems | 247-IL | ||
Protein G | Sigma Aldrich | 19459 | ||
Rec. human ICAM-1/Fc | R&D Systems | 720-IC | ||
Rec. human SDF-1 | R&D Systems | 350-NS |