Optical bottom

Rat ChondroSarcoma cells (RCS) were kindly provided by Dr Claudio Basilico from New York University School of Medicine. Cells were maintained in DMEM high glucose (Gibco) supplemented with 10% hiFBS (Gibco) and 1% Penicillin/Streptomycin/Glutamine (Gibco). For proliferation assay, 2500 cells were plated in black 96-well plate with optical bottom (Nunc). 24h after plating, medium was removed and replaced by assay medium (DMEM high glucose, 1% heat inactivated fetal bovine serum, 1% Penicillin/Streptomycin/Glutamine). Cells were either stimulated for 48h with increasing concentrations of human FGF2 (Peprotech) or 0,3ng/mL human FGF2 and increasing concentrations of Recifercept, both supplemented with 1μg/mL heparin sodium salt. Cell proliferation was assessed by measuring cell nuclei number with CyQuant Direct assay (Thermofisher Scientific). Fluorescence was measured with a Varioskan Lux plate reader.

5000 cells were plated in white 96-well plate with optical bottom (Nunc). Prior to plating, cells were centrifuged twice at 800g, 5 minutes at room temperature and washed with assay medium (RPMI-1640, 10% heat inactivated fetal bovine serum, 1% penicillin/streptomycin/glutamine) to remove of IL-3 and puromycin.
Cells were either stimulated for 72h with increasing concentrations of human FGF1 (Peprotech) or simultaneously with 10ng/mL human FGF1 and increasing concentrations of Recifercept, both supplemented with 10μg/mL heparin sodium salt (Sigma) acting as a co-factor. Cell proliferation was assessed by measuring intracellular ATP concentration with CellTiter Glo assay (Promega). Luminescence readout was measured with a Varioskan Lux plate reader (Thermo Fisher Scientific).

Isolated primary MM cells were labeled with eBioscience^TM Calcein AM Viability Dye (Invitrogen, Waltham, MA, USA) according to the manufacture’s protocol. After labeling, cells were seeded in flat black 96-well plate with optical bottom (Thermo Scientific, Waltham, MA, USA) in density 2 × 10⁴ cells/well and covered with cryopreserved FINM containing WJ-MSCs (2 × 10⁴/well) and NKs (2 × 10⁵/well). After 5 min in 37 °C, cells were covered with 200 μL of full RPMI 1640 medium. After 4 h, plates were spinoculated (5 min, 300 g) and 100 μL of medium from each well was collected for fluorescence (FLUO) measurement. FLUO intensity (excitation 495 nm, emission 515 nm) was measured using Infinite^® F Plex (Tecan, Männedorf, Switzerland, version 3.9.0.1). Because the cell viability of primary MM cells rapidly dropped after cultivation, longer times assessment of cytotoxicity was not possible. Cell viability (%) was evaluated based on fluorescence intensity of Calcein released into the medium.