Synergy htx multi mode

The metabolic activity of the cell cultures was determined using PrestoBlue™ (Invitrogen) assays. We added 10% (v/v) PrestoBlue™ reagent (with respect to the culture medium) to the samples contained in 96-well plates, followed by incubation at 37°C for 1 h. The fluorescence intensity was measured with a microplate reader (Synergy HTX Multi-mode, BioTek, United States) at excitation/emission wavelengths of 530/570 nm. The fluorescence intensities were normalized with respect to the first reading at 12 h.

Standard solutions of sarcosine were prepared at concentration ranging from 1-200 µM (1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100 and 200 µM) using distilled water. The reactions were performed in 96-well microtiter plates (SPL Life Sciences Co., Ltd., Gyeonggi-do, South Korea) and the absorbance was measured by a microplate reader (Synergy HTX multi-mode, BioTek, Winooski, VT, USA). The total volume of each reaction was 200 µL. Reactions were performed by adding 127.5 µL of 50 mM sodium phosphate buffer (pH 7.5), 20 µL of sarcosine solution, 12.5 µL of Amplex Red (0.012 mM), 20 µL of SOX (0.04 U) and 20 µL of HRP (0.3 U). The absorbance (OD570) was immediately measured at 37 °C every 5 min for 30 min. All assays were performed in triplicate. Background absorbance was corrected by subtracting the value of the no-sarcosine control from all sample readings. The optimum pH range was determined in the presence of 50 µM sarcosine using the following buffer: 50 mM citrate for pH 3-5; 50 mM sodium phosphate for pH 6-7.5 and 50 mM Tris-HCl for pH 8-10. All reactions were incubated at 37 °C for 20 min. The optimal incubation time was studied by measuring the reaction every 10 min for 90 min and the reaction was performed in 50 mM sodium phosphate buffer, pH 7.5 in the presence of 100 µM sarcosine.

These assays were performed as described previously [12 (link)]. In the cell viability assay, cells were seeded at 10,000 cells per well in 96-well plates and exposed to AZD4573, zelavespib, and tanespimycin for 72 h. Subsequently, cell lysis was conducted using the CellTiter-Glo Luminescent Cell Viability Assay Reagent, and luminescence was quantified employing the BioTek Synergy HTX Multi-mode microplate reader. For the apoptosis assay, Annexin V-binding was employed. MCL cells were treated separately with the vehicle, AZD4573, zelavespib, and tanespimycin, stained with Annexin-V and propidium iodide, and then subjected to flow cytometric analysis using the Novocyte Flow Cytometer to determine the percentages of Annexin-V positive cells. Data analysis was carried out using NovoExpress or FlowJo10, and each experiment was meticulously repeated at least three times to ensure the reliability of the results, consistent with the procedures outlined in the reference.