Obtain fresh blood specimens from healthy donors .
{"action": "obtain", "output": "fresh blood specimens"}
isolate PBMC by Lymphoprep density gradient medium #####%%%%% output "MACS buffer" are used in line8
{"action": "identify", "output": "MACS buffer", "reagent": ["PBMC", "Lymphoprep density gradient medium"]}
Isolate total CD4<sup>+</sup> T cells using the CD4<sup>+</sup> T cell Isolation Kit as the following steps in brief.
{"action": "isolate", "output": "", "reagent": ["total CD4<sup>+</sup> T cells"], "device": ["CD4<sup>+</sup> T cell Isolation Kit"]}
\(4) Prepare LS column by rinsing with 3 ml of MACS buffer.
{"action": "prepare", "output": "", "container": ["LS column"], "reagent": ["MACS buffer"], "volume": ["3 ml"]}
Wash CD4<sup>+</sup> T cells \(negative fraction) with MACS buffer.
{"action": "wash", "output": "", "reagent": ["CD4<sup>+</sup> T cells (negative fraction)"]}
The purity of Treg cells ranged 95%-98%.

Treat Treg cells with TNF-α \(50 ng/ml) \(100 U/ml) for 24 hs.
{"action": "treat", "output": "", "reagent": ["Treg cells"], "time": ["24 hs"]}
leave them in medium alone \(control) in 96-well U-bottom plates \(0.5-1.0×10<sup>5</sup> cells per well) in the presence of IL-2
{"action": "leave", "output": "", "concentration": ["0.5-1.0\u00d710^5 cells per well"]}
Wash the resulting Treg cells twice with culture medium, . #####%%%%% output "the resulting Treg cells" are used in line20
{"action": "rinse", "output": "the resulting Treg cells", "reagent": ["culture medium"]}
count the cells #####%%%%% output "CFSE" are used in line22
{"action": "estimate", "output": "CFSE", "container": ["the cells"], "volume": [""], "concentration": [""], "reagent": ["the resulting Treg cells"]}
Label Teff cells with CFSE at 2 µM, 37°C for 8 min, then wash for three times with culture medium.
{"action": "rinse", "output": "", "reagent": ["CFSE"]}
Maintain the culture in the presence of 2×10<sup>3</sup> anti-CD3/CD28 beads for 4 days.
{"action": "maintain", "output": "", "concentration": ["2\u00d710<[sup>3</sup>] anti-CD3/CD28 beads"], "time": ["4 days"], "temperature": [""]}
Maintain the culture in the presence of 2×10<sup>3</sup> anti-CD3/CD28 beads for 4 days.
{"action": "maintain", "output": "", "concentration": ["2\u00d710<[sup>3</sup>] anti-CD3/CD28 beads"], "time": ["4 days"], "temperature": [""]}
Express the results as percent inhibition: \(1 − \(experimental CFSE dilution/control CFSE dilution)) #####%%%%% output "percent inhibition" are not used
{"action": "connect", "output": "percent inhibition", "device": [""]}