Primaria 24 well plates

PMPs were plated in Primaria 24-well plates (Corning, 353847) at a density of 100,000 cells per well for microglia differentiation. iMGs were pulse-labeled with 12 µg/mL green DQ-BSA reagent (Invitrogen, D12050) for 1 h, washed with DPBS, and subjected to live-cell imaging after adding fresh iMG media (Table 1). As a negative control, cells were treated with 200 nM of bafilomycin A for 30 min before the assay. Twenty phase and green fluorescence images were acquired per well from two technical replicates at 20× using the Cytation 5 cell imaging reader (Biotek). Images were acquired immediately after washing and every hour for four hours. Images were analyzed to obtain total DQ-BSA intensity values and the number of cells by counting the cell nuclei from all the acquired fields of view with Gen5 software. Total intensity values were divided normalized to the number of cells and average value of the technical replicates are reported.

Human OA chondrocytes were plated directly after isolation in culture medium at 340 mOsm (= unchanged osmolarity) or in culture medium with an osmolarity adjusted to 380, 420 or 460 mOsm in PRIMARIA™ 24 well plates (Corning, New York, NY, USA) at 240,000 cells/well. The media were renewed after three or four days and cells were cultured for six days. A total of five donors were used and for each donor a minimum of six cell cultures were realized for each condition; three were used for determination of the cell concentration (resulting in a minimum of n = 3 technical replicates per donors) and the three others for gene expression analysis (resulting in n = 3 technical replicates per donors or n = 2 when RNA isolation was not successful in one of the samples). The GAG (n = 3–6 per donor) and cytokine concentrations (n = 3–6 per donor) were analyzed in the medium.