Source: Juvet, S. C. et al., Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry. J. Vis. Exp. (2017)
This video demonstrates a method for assessing alloreactivity in mixed T cell populations by observing interactions with alloantigen-carrying dendritic cells, leading to the formation of immunological synapses. Differential staining and quantification of synapse-containing doublets via flow cytometry reveal T cell alloreactivity.
1. Prepare Reagents and Materials Required
2. Prepare Antigen-presenting Cells (APCs)
NOTE: In theory, any APC population could be examined with this method. Immature mouse bone marrow-derived dendritic cells (DCs) as APCs were sued in this case. Many protocols exist for generating these cells. Briefly, the following protocol was used.
3. Prepare T Cells
4. Co-incubate T Cells and DCs
5. Fix Cells in Plate
6. Stain Cells
7. Acquire Data
Figure 1. Gating Strategy Used to Identify Alloreactive Immune Synapses. A. In-focus events are gated from all events by reviewing cell images based on the root mean square of the rate of change of the image intensity profile (Gradient RMS) using the brightfield channel (Channel 4, Ch04), as described in the text. B. Among in-focus events, doublets are distinguished from single cells by plotting the aspect ratio versus area for the brightfield channel. Single cells are clustered close to the aspect ratio of 1 and have a smaller area, while doublets are close to 0.5 and have a larger area. C. Fluorescence intensity of the APC (in this case, a dendritic cell [DC] marker, CD11c) is then plotted against the fluorescence intensity of the T-cell marker (in this case, CD90.2), and double-positive events are gated. Borders of the gate can be refined by reviewing images of events near the borders. D. T-APC doublets are then refined so that they contain only one APC by plotting the aspect ratio versus the area of the APC marker (CD11c, Ch02). E. These single-APC doublets are then refined so that they contain only one T cell by plotting the aspect ratio versus the area of the T-cell marker (CD90.2, Ch06). F. Finally, events containing only two nuclei are selected by plotting a histogram of the spot count on the nuclear stain channel (7-AAD, Ch05) and gating events that contain only 2 7-AAD-positive spots (i.e., nuclei). The events in this gate are analyzed for membrane contact and synapse formation. The data were analyzed in a blinded fashion with respect to treatment assignment and are from a previously published experiment.
The authors have nothing to disclose.
Phosphate-buffered saline | Various | Varies | |
Ethylenediamenetetraacetic acid, 0.5M solution | Thermo Fisher Scientific | AM9260G | |
Triton X-100 nonionic detergent | Sigma-Aldrich | X100 | |
Beta-mercaptoethanol | Sigma-Aldrich | M3148 | |
Dimethyl sulfoxide | Sigma-Aldrich | D8418 | |
Formaldehyde | Sigma-Aldrich | F1635 | Solution is 37% formaldehyde and so must be diluted 25 times for 1.5% solution |
Cell strainers, 70 μm pore size | Fisher Scientific | 08-771-2 | |
Phalloidin-fluorescein isothiocyanate | Sigma-Aldrich | P5282 | |
7-aminoactinomycin D | Thermo Fisher Scientific | A1310 | Reconstitute in DMSO |
Allophycocyanin-conjugated anti-mouse CD90.2 | eBioscience | 17-0902 | |
Pacific blue-conjugated anti-mouse CD11b | eBioscience | 48-0112 | Pacific blue has been replaced by eFluor 450 |
Biotinylated anti-mouse CD3 | eBioscience | 13-0032 | |
Biotinylated anti-mouse MHC class II | eBioscience | 13-5321 | |
Biotinylated anti-mouse B220 | eBioscience | 13-0452 | |
Biotinylated anti-mouse CD8 | eBioscience | 13-0081 | |
Biotinylated anti-mouse CD19 | eBioscience | 13-0193 | |
Anti-biotin microbeads | Miltenyi Biotec | 130-090-485 | |
LS columns | Miltenyi Biotec | 130-042-401 | |
MidiMACS magnetic cell separator | MIltenyi Biotec | 130-042-302 | |
Recombinant mouse GM-CSF | Peprotech | 315-03 | |
Recombinant human TGFβ1 | Peprotech | 100-21 | Human TGFβ1 has activity on mouse cells |
Amnis ImageStream X Mark II | Amnis/EMD Millipore | N/A | Imaging flow cytometer; details available at http://www.emdmillipore.com/ |
IDEAS Software | Amnis/EMD Millipore | N/A | Free download (registration required): https://www.amnis.com/index.php/page/Display/login%20%20 |
Cell culture medium | Various | Varies | |
Fetal bovine serum | Various | Varies | |
Cell culture plates | Various | Varies |