BrdU cell proliferation assays

The proliferation of PBMC and TF-1 was evaluated using BrdU Cell Proliferation Assay (Calbiochem).

The freshly isolated PBMC were seeded in a 96-well V-bottom plate (25 × 10³ cells in 200 µl of the culture medium/well) and cultured for 24 hours. Next, the cells were incubated for 30 minutes with cefazolin at concentrations of 100 µM, 200 µM and 400 µM. Next, 20 ng/ml IL-2 or 5 ng/ml IL-15 was added and the cells were incubated for 72 hours in 37 °C, 5% CO₂. For the last 24 hours of incubation bromodeoxyuridine (BrdU) was added to the culture medium at the concentration recommended by the manufacturer. Then, the cells were centrifuged (160 x g, 10 minutes) and fixed. Further experimental steps were performed according to the manufacturer’s protocol. The experiment was performed using cells isolated from three blood donors, each in five technical replicates.

TF-1 cells were starved in a RPMI 1640 with 10% FBS and 1% AA solution for 24 hours. Then 40 × 10³ cells were seeded in a 96-well V-bottom plate with or without cefazolin at concentrations of 100 µM, 200 µM and 400 µM and incubated for 30 minutes in 150 µl of RPMI 1640 medium containing 10% FBS and 1% AA solution. Next, 10 ng/ml IL-4 (Invitrogen, Gibco) was added and the cells were incubated for 72 hours in 37 °C, 5% CO₂. For the last 24 hours of incubation BrdU was added to the culture medium at the concentration recommended by the manufacturer. Then, the cells were centrifuged (1,000 x g, 10 minutes) and fixed. Further experimental steps were performed according to the manufacturer’s protocol. The experiment was performed three times, each in five technical replicates.

The description of two additional proliferation assays, CFSE cell proliferation assay for PBMC treated with IL-2 and IL-15 and MTT cell cytotoxicity assay for TF-1 stimulated with IL-4, is provided in Supplementary Material and Methods.