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
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
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|
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 |
|Treg Suppression Inspector |
(1×107 MACSiBead Particles/mL)
|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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