Measuring Alloreactivity in a Mixed Population of T Cells
Published: January 31, 2024
Abstract
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.
Protocol
1. Prepare Reagents and Materials Required Prepare phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) ("wash buffer"). Prepare PBS with 2% fetal calf serum (FCS) containing 0.1% nonionic detergent ("perm-wash buffer"; see the Table of Materials). Prepare PBS with 1.5% formaldehyde. NOTE: Formaldehyde is corrosive and potentially carcinogenic and must be handled while wearing appropriate personal protective equipment. Prepar…
Representative Results
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 distinguishe…
Declarações
The authors have nothing to disclose.
Materials
| 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| Pacific blue-conjugated anti-mouse CD11b | eBioscience | 48-0112 | Pacific blue has been replaced by eFluor 450 |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 |
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Citar este artigo
Measuring Alloreactivity in a Mixed Population of T Cells. J. Vis. Exp. (Pending Publication), e21913, doi: (2024).