Measuring MDSC Migration Inhibition

Cell migration assays were performed using FluoroBlok™ 96-Multiwell Insert Plates with 3.0 µm High Density PET Membrane (#351161, Thermo Fisher Scientific, Waltham, MA, USA) as described before (10 (link)). The CD11b⁺Gr1⁺ MDSCs were isolated using the EasySep™ Mouse Myeloid-Derived Suppressor Cell Isolation kit (#19867, Stemcell Technologies, Cambridge, MA, USA) according to the manufacturer’s instructions. The isolated cells were prelabeled with 5 μM of calcein AM (#564061, BD Biosciences, San Jose, CA, USA) and then were seeded in the upper chamber at the density of 1 × 10⁵/50 µl/insert. Normal MLFs were cultured with or without (control) TGFβ treatment (10 ng/ml) for 24 h. The media containing residual TGFβ were removed, and after washing, fresh media were added. Conditioned media (CMs) were collected after an additional 48 h. The control or TGFβ-treated MLF CMs were incubated with B7H3 blocking antibody (15 µg/ml) or isotype IgG and then added to the lower chamber of the plates. The plates were incubated for 18 h followed by fluorescence measurement using SpectraMax Gemini EM Microplate Reader (Molecular Devices, San Jose, CA, USA).