Synergy h1 automatic plate reader

Cells were scraped from the culture plates in phosphatase extraction buffer containing 20 mM imidazole-HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0, and with 10 µg/mL each of aprotinin, leupeptin, antipain, soybean trypsin inhibitor, 1 mM benzamidine, and 1 mM PMSF, and then were sonicated for 10 s and centrifuged at 2000× g for 5 min. The supernatants were used for phosphatase activity assays per the manufacturer’s protocol (Sigma-Millipore, Saint Louise, MO, USA). In brief, 500 µg of lysate were immunoprecipitated with anti-PP2A, C subunit (clone 1D6) and the protein immunocomplexes were collected with a Protein A agarose slurry. The PP2A activity was assessed by dephosphorylation of a phosphor-peptide substrate and measured by a Malachite Green phosphate detection solution at 650 nm with a BioTek Synergy H1 automatic plate reader (Winooski, VT, USA).

For the 3D co-cultures of bone cells (MC4 pre-osteoblasts or MLOA5 osteocytes) and MM cells (MM.1S-gLUC), an equal parts mixture of basement membrane extract (BME, Culturex Trevigen #3433-005) and cells in 10% FBS α-MEM were added to 96-well plates and monitored for up to 72 h. A 1:5 ratio of bone cells to MM was used for the co-culture groups [42 (link),43 (link)]. BME was thawed overnight at 4 °C. Cells of interest were counted and pelleted via centrifugation. The cell pellets were resuspended in 50 μL of 10% FBS α-MEM, then 50 μL of thawed BME was added to the cells. The 100 μL cells/BME mixture was then added to wells of a 96-well plate and incubated at 37 °C for 30 min. After incubation, 125 μL of 10% FBS α-MEM (bone cells alone), 10% FBS RPMI (MM cell alone), or a 1:1 mixture of 10% FBS α-MEM and 10% FBS RPMI (co-cultures)—with or without SK1-I inhibitor (10 μM)—was added to the wells. The cultures were checked daily via microscopy, and MM viability and cell number were analyzed by luminometry using a Gaussia luciferase assay (New England Biolabs, #E3308), per the manufacturer’s protocol, and a BioTek Synergy H1 automatic plate reader (Winooski, VT, USA).