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As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is committed to providing our customers around the world with the highest quality products. In addition to direct selling in more than 20 countries in North America, Europe and Asia/Pacific, Miltenyi Biotec also provides support for our customers through an extensive distributor network covering dozens of additional countries.
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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.
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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 |
Note:
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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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