2 After mashing the spleens, pass the cells through the nylon strainer in the medium. #####%%%%% output "the cells" are not used
{"action": "pass", "output": "the cells", "device": ["the nylon strainer"], "reagent": ["medium"]}
3 After washing the cells with PBS/BSA, osmotically lyse the erythrocytes . #####%%%%% output "FITC-conjugated anti-CD4 CD25" are used in line6
{"action": "detect", "output": "FITC-conjugated anti-CD4 CD25", "reagent": ["PBS/BSA"]}
incubate the single cell suspensions with FITC-conjugated anti-CD4 CD25 for 30 minutes on ice #####%%%%% output "anti-FITC microbeads" are used in line10
{"action": "incubate", "output": "anti-FITC microbeads", "reagent": ["FITC-conjugated anti-CD4 CD25"], "temperature": ["ice"], "time": [""]}
\(each 2&#xB5;g/ml concentration in 3ml volume of PBS/BSA) 4  After washing in 40ml of PBS/BSA, resuspend the cells in 2ml of PBS/BSA .
{"action": "resuspend", "output": "", "reagent": ["cells"], "volume": ["2 ml"], "solution": ["PBS/BSA"]}
incubate with 120&#xB5;l of anti-FITC microbeads for 15 minutes in the refrigerator #####%%%%% output "release reagent (available" are used in line18
{"action": "incubate", "output": "release reagent", "reagent": ["anti-FITC microbeads"], "temperature": ["the refrigerator"], "time": [""]}
This is followed by washing with PBS/BSA.

5) Purify CD4+ T cells by magnetic isolation using the Auto MACS sorter \(Miltenyi Biotec) using POSSELD2 program.
{"action": "purify", "output": "", "device": ["Auto MACS sorter"]}
Every incubation step is followed by PBS/BSA washing.

6  Release the Anti FITC beads by incubating with release reagent \(available in the multisort kit) followed by the depletion of cell that are bound to beads. #####%%%%% output "CD4+CD25+ Tregs" are used in line22
{"action": "release", "output": "CD4+CD25+ Tregs", "reagent": ["release reagent (available"], "device": ["the multisort kit"]}
This is performed by the DEPLETE program in the sorter.

7  For isolation of CD4+CD25+ Tregs, after releasing the beads, incubate the purified CD4T cell suspension \(500&#xB5;l) with 2.5&#xB5;l of &#x3B1;-biotin microbeads followed by POSSELD2 separation using the Auto MACS. #####%%%%% output "the purified CD4T cell suspension" are not used
{"action": "incubate", "output": "the purified CD4T cell suspension", "reagent": ["α-biotin microbeads"], "volume": ["500 µl"]}
The negative fractions were depleted of CD25cells to obtain CD4+CD25&#x2013; . #####%%%%% output "CD4+CD25&#x2013;" are used in line32
{"action": "recover", "output": "CD4+CD25&#x2013;", "reagent": ["CD25cells"]}
3&#xD7;10<sup>4</sup> irradiated \(3000 Rad) T cell-depleted splenocytes can be added as APCs in the co-culture.
{"action": "add", "output": "", "reagent": ["3×10^4 irradiated (3000 Rad) T cell-depleted splenocytes"]}
Use Treg Tcon cells in the co-culture with responders directly \(fresh) IL-2 \(100U/ml) for 2-3 days. #####%%%%% output "CFSE+ responders" are used in line30
{"action": "use", "output": "CFSE+ responders", "reagent": ["Treg Tcon cells", "responders"]}
Cell death analyses of CFSE+ responders should be performed based on forward scatter . #####%%%%% output "forward scatter" are not used
{"action": "perform", "output": "forward scatter", "reagent": ["CFSE+ responders"]}
Assess cell death with events acquired at constant time, in order to count the live events in flow cytometry analyses. #####%%%%% output "cell death" are not used
{"action": "evaluate", "output": "cell death", "device": ["cytometry analyses"], "reagent": ["CD4+CD25&#x2013;"]}