Biotek synergy ht multidetection microplate reader

Total RNA was extracted from transfected SW480 and SW620 cells using RNAzol^® RT (Sigma Aldrich, R4533) following the manufacturer’s protocol. Isolated RNA was resuspended in nuclease-free water and quantified using a BioTeK Synergy HT MultiDetection Microplate Reader™ (BioTek, USA). Following that step, RNA reverse transcription was performed in an AriaMx Real-time PCR System (Agilent Technologies, Santa Clara, CA, USA) using the 5x All-In-One RT MasterMix kit (Applied Biological Materials, Bellingham, WA, USA), and product cDNA was diluted into a final concentration of 20 ng/μL.
Afterward, 40 ng of cDNA was used for the analysis of target genes by real-time qPCR in an AriaMx Real-time PCR System (Agilent Technologies, Santa Clara, CA, USA) using the Brilliant II SYBR Green qPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA) following the standard manufacturer’s protocol. Amplification curves for each qPCR reaction were analyzed using the Agilent AriaMx Software 1.71, and the specificity of PCR products was controlled by analyzing the respective melting curves and temperatures. Relative expression of target genes was calculated by normalizing against the housekeeping gene pumilio RNA-binding family member 1 (PUM1) using the ΔΔCt method. Primer pair sequences are listed in Supplementary Table S1.

We seeded 5 × 10³ cells per well in 96 well plates for MTT assays. Cells were cultured until ∼60% confluence and then transfected with miR-92a-3p mimics, inhibitors, and control oligonucleotides, according to the protocol previously defined. Non-transfected cells were used as an additional control for this assay. After 6 h of transfection, 100 μL of Opti-MEM™ culture medium with different concentrations of 5-FU/L-OHP was added to each well until reaching the final concentrations of 0 μM, 25/5 μM, 50/10 μM, 75/15 μM, 100/20 μM, 150/30 μM, 200/40 μM, and 250/50 μM per well (total volume = 200 μL). Treated cells were kept at 37 °C with 5% CO₂ for 24 h. Following that time, we discarded the FOLFOX-Opti-MEM™ culture medium and added 100 μL per well of a solution of MTT 150 μg/mL (Sigma-Aldrich, St Louis, MO, 23 USA) in Locke’s buffer and incubated for 4 h. After this, we discarded the MTT-Locke’s solution and solubilized the resulting formazan crystals in 100 μL DMSO. Absorbancy of the resulting solution was measured at 570 nM using a BioTeK Synergy HT MultiDetection Microplate Reader™ (BioTek, USA).

Saos2 cells [30 ] were purchased from Cedarlane (Supplier ATCC, Burlington, ON, Canada). HAM-F12, 10× Trypsin, PBS, fetal bovine serum (FBS), antibiotic-antimycotic and flasks were purchased from Lonza (Mississauga, ON, Canada). Rutin trihydrate (≥97%) and 3-(4,5-dimethy-2-lthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide were purchased from Alfa Aesar (Reston, VA, USA) and hyperoside (≥98%) from Aktin Chemicals (Sichuan, China). Human Milliplex bone magnetic bead panels for bone metabolism were purchased from EMD Millipore (Billerica, MA, USA) and read by xPONENT software on Magpix Luminex (Austin, TX, USA). Optical density (OD) measurements were analyzed using a BIO-TEK Synergy HT Multi-Detection Microplate Reader (Winooski, VT, USA). Other chemicals were purchased from Sigma-Aldrich (Oakville, ON, USA).
Cells were maintained and differentiated as previously described [31 (link)]. Following one week of differentiation in plates, differentiation cell media was supplemented with 0.01 M β-glycerophosphate (day 8) until day 21 (Media 3) in addition to either vehicle (0.1% DMSO) or flavonoid dissolved in DMSO to 0.1%.
For all experiments, a DMSO control was compared to flavonoid treated cells. All experiments were normalized to cell count using Trypan Blue staining.