T Cell Proliferation Assay with LrS235 and LrGusA

Peripheral blood MNC were loaded with 5 µM CellTrace Violet (Invitrogen) according to the manufacturer’s instructions (58 (link)) and incubated for 30 min with sterile-filtered supernatants from LrS235 or LrGusA, buffered to pH 7.4 and diluted 1/10 in tissue culture media, before the addition of anti-CD3 antibodies to stimulate T lymphocytes (clone OKT3, 1 ng/ml, 037-85, Thermo Fisher). Seven days later, cells were stained with anti-CD3 antibodies conjugated to phycoerythrin (BioLegend 300308, lot B209105) and anti-CCR7 antibodies conjugated to fluorescein (R&D Systems, FAB197F, lot LEU1615081), and the dilution of CellTrace Violet in CD3⁺CCR7⁻ T_EM cells and CD3⁺CCR7⁺ naive/T_CM cells was measured by flow cytometry on a BD FACSCantoII as quantification of cell proliferation. Data were analyzed with FlowJo.