In vitro human regulatory T cell suppression assay
This application protocol describes every step from human Treg and Tresp cell isolation, to their co-cultivation in an in vitro suppression assay using the Treg Suppression Inspector, and flow cytometry analysis.
1× PBS (pH 7.2), 0.5% BSA, 2 mM EDTA
or
6× 1.5 L autoMACS® Running Buffer – MACS® Separation Buffer
The suppression medium can be stored for up to 7 days under sterile conditions at 4 °C.
500 mL TexMACS™ Medium
+ 5% human AB serum
+ 1% PenStrep
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Number of Treg cells, Tresp cells and Treg Suppression Inspector (MACSiBead Particles) per well of a 96-well flat-bottom plate.
Ratio Tresp:Treg | Tresp | Treg | Treg Suppression Inspector |
---|---|---|---|
1:0 | 5×104 | - | 5×104 |
1:1 | 5×104 | 5×104 | 1×105 |
2:1 | 5×104 | 2.5×104 | 7.5×104 |
4:1 | 5×104 | 1.3×104 | 6.3×104 |
8:1 | 5×104 | 6×103 | 5.6×104 |
0:1 | – | 5×104 | 5×104 |
1:0 | 5×104 | – | – |
0:1 | – | 5×104 | – |
Total | 3×105 | 2×105 | 4×105 |
Total triplicates | 9×105 | 6×105 | 1.2×106 |
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Pipetting scheme for one assay with a total volume of 210 µL of cell suspension per well with a concentration of 5×105 cells/mL.
Ratio Tresp:Treg | Tresp (5×105 cells/mL) | Treg (5×10⁵ cells/mL) | Treg Suppression Inspector (1×107 MACSiBead Particles/mL) | Culture medium |
---|---|---|---|---|
1:0 | 100 µL | – | 5 µL | 105 µL |
1:1 | 100 µL | 100 µL | 10 µL | – |
2:1 | 100 µL | 50 µL | 7.5 µL | 53 µL |
4:1 | 100 µL | 25 µL | 6.5 µL | 79 µL |
8:1 | 100 µL | 12.5 µL | 6 µL | 92 µL |
0:1 | – | 100 µL | 5 µL | 105 µL |
1:0 | 100 µL | – | – | 110 µL |
0:1 | – | 100 µL | – | 110 µL |
Total volume | 600 µL | 387.5 µL | 40 µL | 654 µL |
Total volume triplicates | 1800 µL | 1200 µL | 120 µL | ~ 2 mL |
Incubate the suppression assay at 37 °C and 5–7% CO₂ for 5 days.
▲ Note: If a proliferation dye (e.g. CellTrace or CFSE) was used, refer to "Flow cytometry analysis" for detailed description of final flow cytometry analysis.
▲ Note: If 3H-thymidine was used: after 4 days add the appropriate volume of 3H-thymidine to each well and incubate at 37 °C and 5–7% CO₂ for 16 hours. Measure 3H-thymidine incorporation, e.g., by using a liquid scintillation counter.
The data obtained by flow cytometry analysis can be summarized in a graphic as depicted in the figure below.
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