Cyquant

YRS, heat-denatured YRS, CCL7 (all 50 nm), or PBS in RPMI 1640 medium, 20 mm HEPES, 0.1% bovine serum albumin (BSA), 10 μg/ml polymyxin B (chemotaxis buffer) was added to the lower wells of chemotaxis chambers (Neuroprobe). THP1 monocytes, synchronized by serum starvation, were resuspended in chemotaxis buffer (1 × 10⁶ cells/ml), and 2 × 10⁵ cells were placed in the top wells separated from the lower chamber by a 5-μm membrane. After incubation for 90 min at 37 °C, cells were harvested from the lower well and quantified using CyQUANT® (Thermo Fisher Scientific). CCL7 was synthesized as described previously (12 (link)).

Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using the extracellular flux analyser XFe96 from Agilent Seahorse Bioscience as previously optimized for PCa cells [10 (link), 12 (link), 19 (link)]. Cells were plated at 10 000 cells/well confluency in a 96‐well Seahorse microplate in phenol red‐free RPMI medium supplemented with 5% CSS. After 48 h, cells were treated with androgens and/or IDH1 inhibitors. After another 48 h, media was changed for Seahorse XF RPMI Medium supplemented with 10 mm glucose, 2 mm glutamine, 1 mm sodium pyruvate, and 100 U·mL⁻¹ penicillin–100 μg·mL⁻¹ streptomycin. The microplate was then transferred to a CO₂‐free incubator at 37 °C for 1 h. After equilibration, the microplate was inserted into the XFe96 instrument. Basal respiration as well as respiration during a mitochondrial stress test were measured by three consecutive measurements of OCR and ECAR before and after injection of oligomycin (Sigma), FCCP (Santa Cruz), and antimycin A (Sigma) + rotenone (Sigma). For normalization, cell counting was performed using CyQUANT (Thermo Fisher Scientific) assay as described previously [25 ].

LDH Release was measured using a CyQuant (ThermoFisher, Dublin, Ireland) LDH cytotoxicity kit following the manufacturer’s instructions. To summarize, after cell treatment, the cell supernatant was mixed with LDH substrate and assay buffer at a 1:1:1 ratio and incubated at room temperature for 30 min on a plate rocker covered with foil. After incubation, the same volume of stop solution as used for every other reagent was added to each well and the absorbance was measured at 490/680 nm using a Clariostar plate reader.

DNA content (Cyquant, ThermoFisher Scientific, Waltham, MA, USA) and Oil Red O (Sigma, St. Louis, MO, USA) assays were preformed according to manufacturers’ protocols (n = 3). Cells were imaged using an IX-81 microscope (Olympus, Center Valley, PA, USA). Cells stained with Oil Red O were imaged using a VHX digital microscope (Keyence Corp., Osaka, Japan). For quantitative determination of Oil Red O staining, the stain was extracted using isopropanol and absorbance was measured at 405 nm using ELX-800 absorbance plate reader (Winooski, VT, USA). To measure DNA content, cells were first removed from the scaffolds using a collagenase I solution (Sigma) and then lysed using a Branson Digital Sonifier 450 (Danbury, CT, USA).

Myeloma free light chains were purified from the urine of patients who had multiple myeloma, light chain proteinuria, and clinical evidence of significant renal damage, using ammonium sulfate precipitation and Sephadex chromatography as described previously⁴³ . The purity and identity of the myeloma light chains were confirmed by SDS-PAGE and Western blotting and all specimens were determined to be endotoxin-free by Liumulus ameboecyte assay. Myeloma light chains were stored in lyophilized form until dissolved in tissue culture media and sterile-filtered before addition to cells. Myeloma light chains from six donors were evaluated, in serial dilutions, for their toxic effects on renal cells, in static adherent cultures using 96-well plate format. Cell proliferation was measured with CyQUANT (ThermoFisher) using manufacturer's protocol. The isolation and use of the myeloma light chains was approved by the IRB at the Tulane Office of Human Research Protection (IRB reference no. 848169). The experiments were performed in accordance with relevant guidelines and regulations, informed consent was obtained from all participants and/or their legal guardians, and all protected health information was deidentified.

Cell growth was assessed using the MTT (MilliporeSigma) or Cyquant (Thermo Fisher Scientific) assays following manufacturer protocols. The Cyquant assay was used for experiments involving TMZ. For the MTT assay, cells were plated (U251, 4,000 cells; GBM10, 5,000 cells; GBM12, 5,000 cells; GBM14, 3,000 cells per well) in triplicate in 96-well plates and allowed to grow for 5–8 days. For the Cyquant assay, U251 cells were plated (500 cells per well) and allowed to grow for 24 hours prior to 5- to 6-day treatment with indicated concentrations of TMZ. For the radiation experiments, U251 and T98G cells expressing shRNAs were plated (4,000 cells per well), subjected to radiation a day later using a RAD-160 Biological Irradiator (Precision X-Ray), and cell viability was assessed by the MTT assay 8 days after radiation. Synergy analysis was performed using the Calcusyn software (Biosoft) with the Chou-Talalay method. CI was used to describe synergistic (CI < 1), antagonistic (CI > 1), or additive (CI = 1) drug interactions. Heatmaps were generated using the GraphPad PRISM software.