The following protocol describes a basic type of in vitro Treg suppression assay where Treg function is measured in the absence of antigen-presenting cells (APCs). In this protocol, activation is mediated by anti-CD3 + anti-CD28 coated beads and, therefore, includes only two cell types, the target Tconv and test Tregs. In this protocol, the experiment is setup in a 96-well round-bottom plate in a total volume of 200 μl. All reagents are prepared at four times their desired final concentration and added to assay in 50 μl such that in the total volume of 200 μl, their concentration will be correct. See Fig. 1a for a 96-well plate layout (see Note 2).

Purify Tregs and Tconv from desired source (see Subheading 3.8).

Count Tregs and Tconv and adjust in T-cell culture medium (see Subheading 2.1) to 2.5 × 105/ml and 5 × 105/ml, respectively.

In round-bottom 96-well plate, add 50 μl culture media to wells 1–11 (see Fig. 1b).

Add 100 μl Treg to well 12.

Mix Tregs thoroughly with a pipet and titrate 50 μl of Tregs into well 11 to generate a twofold dilution. For multiple Treg populations, use a multichannel pipet to titrate multiple wells at the same time.

Repeat mixing and titration into successive wells, 50 μl at a time, leaving the well 6 with no Treg to determine maximum proliferation of Tconv.

Add 50 μl Tconv cells to all wells.

Add 100 μl anti-CD3/CD28-coated sulfate latex beads to all wells (see Subheading 3.9).

Incubate plate at 37°C, 5% CO2 for 72 h.

Pulse plates with 0.1 μCi [3H]-thymidine (<!> – Caution: Radioactive material. Institutional approval to handle radioactive materials is required) per well 8 h prior to completion of experiment.

Harvest cultures with a commercial cell harvester and determine counts per minute (cpm) with a direct beta counter (see Notes 3 and 4).