Protocol to analyze transmigration of human cytotoxic T cells under physiological flow conditions in vitro

Abstract

This protocol presents an assay for transmigration analysis of human cytotoxic T cells (CTL) under physiological flow in vitro. We describe detailed analysis steps of human CTL behavior, from adhesion to diapedesis, using live cell imaging which cannot be achieved by in vivo imaging. The flow system is made of 2D plastic surfaces covered by an endothelial monolayer limiting the system but allows for quantitative analysis of CTL behavior with high modifiability. For complete details on the use and execution of this protocol, please refer to Schoppmeyer et al. (2022).

Keywords: Cell Biology; Immunology; Microscopy.

Graphical abstract

Figure 1

Example transmigration analysis of human CTL on 20 h TNFα-activated HUVEC (A and B) A HUVEC monolayer in a μ-channel VI 0.4 is shown with and without inflammatory stimulation for 20 h. White spots in a are bypassing CTL. Images were taken after cell injection but before cell adhesion occurred. (C–F) example results of a control experiment prepared as described in this protocol. Note the different morphology of the HUVEC cells after inflammatory stimulation. For A see Methods video S1 and for B see Methods video S2 (related to: Image analysis and quantification of transmigration parameters). Error bars are standard deviation calculated by graphpad prism 9. Scale bar represents 100 µm (A and B at same magnification).

Figure 2

Morphologies of CTL contacting inflamed HUVEC monolayers (A) The morphologies usually observed for CTL directly after adhesion to inflamed HUVEC monolayers from flow are shown. Extended is a rare case where CTL detachment seems affected. If this occurs in high frequency in controls troubleshoot CTL and HUVEC culture conditions and inflammation efficiency. The spreading phenotype can often be observed for transcellular transmigrating CTL. In those cases the spreading is quickly followed by a depolarization (returns to rounded cell morphology, examples in Methods video S3). The migratory phenotype results in apical migration of adhering CTL and represents the classical CTL morphology when migrating in a tissue matrix or 2D systems (Schoppmeyer et al., 2017; Zhou et al., 2018). This morphology precedes paracellular events but CTL also adapt this morphology and migrate apically without transmigrating. Scale bar, 10 µm. (B) When transmigrated CTL change contrast to a homogenous dark appearance. Examples are indicated by circles. Note that in still images transmigrated CTL are difficult to identify but very obvious in image series. Thus we always recommend recording a series over 20 min at as low intervals as 1 up to 10 s (see Methods videos S4 and S5). Scale bar, 10 µm. (C) shows the most common parameters important for general analysis: non-polarized (chemotactic stimulation), apically migrating (polarized) and transmigrated CTL. Red asterisks mark CTL (related to: Image analysis and quantification of transmigration parameters).

Figure 3

Flow channel setup (A) In (A) the complete tubing is shown as required for setting up the flow system. (B) Placement of the lid when using several channels for flow experiments. Black outline indicates shifted lid position. This avoids evaporation of the medium from the channels during recordings. The use of a humidifier in the microscope chamber is still recommended. (C) to properly attach the Luer locks from the flow tubing to the channel without trapping air bubbles in the inlets, apply medium to one inlet until a meniscus forms on both inlets (water was used for demonstrative purposes and colored blue for visibility). (D) Successful connected Luer connectors without air inclusion. (E–G) Coating solutions and cell suspension loading is performed by tilting the pipet tip towards the channel inlet at the bottom of the loading chamber (E). Add coating solution or cell suspension in one quick eject (F). Successful loading or seeding with homogenous distribution of the liquid and no air inclusions (G) (related to: Preparation of the flow system).