Whole-cell uptake experiments were performed following published protocols.20 (link) Briefly, 1×106 HCT116N or HCT116O cells were plated into 6-well tissue culture treated plates and allowed to adhere for 24 h. Media was aspirated from the cells and fresh media containing a metalloinsertor was added to each well. Cells were allowed to incubate for an additional 0.5–24 h with the Rh-containing media. After incubation, media was aspirated and the cells were rinsed with PBS (phosphate buffered saline, pH 7.4) to remove surface rhodium. Cells were lysed directly in the well using 1 mL of 1% SDS solution. These samples were transferred to microcentrifuge tubes and sonicated for 10 s at 20% amplitude on a Qsonica Ultrasonic sonicator. Cell lysate was combined with an equal volume 2% HNO3. This solution was analyzed for Rh content on an Agilent 8800 Triple Quadrupole ICP-MS and the concentration of Rh in each sample was determined by comparison to a standard curve (ranging from 1–100 ppb Rh) and normalized using the protein content of each sample. The protein content of each sample was determined using a Pierce BCA assay, following the manufacturer’s instructions.
Ultrasonic sonicator
Manufactured by Qsonica
Sourced in United States
The Ultrasonic Sonicator is a lab equipment used for the application of ultrasonic energy. It generates high-frequency sound waves to disrupt and homogenize samples, such as cells, tissues, or particles, in a liquid medium.
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Lab products found in correlation
3 protocols using ultrasonic sonicator
1
Quantifying Cellular Rhodium Uptake
2
Protein and Cytokine Profiling of Hippocampus
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The total protein content of the hippocampal samples was determined using an assay that was a reducing agent compatible as well as a detergent compatible (Bio-Rad Laboratories, Hercules, CA, USA). The samples were prepared for analysis by adding ice-cold phosphate-buffered saline (0.01 M, pH = 7.4) and volume added based on the sample weight. The samples were then placed in ice and homogenized to disrupt the cell wall and release the cell contents using an ultrasonic sonicator (Qsonica, Newtown, CT, USA). The homogenates were then centrifuged for 5 minutes at 5000g to obtain the supernate. The supernate was transferred to new Eppendorf tubes. The concentration of IL-1ra was measured using a commercially available Rat IL-1ra enzyme-linked immunosorbent assay kit (Elabscience Biotechnology, Wuhan, China). The concentration of IL-1β, TNF-α, IL-6, and IL-10 was measured using a commercially available Bio-Plex Multiplex Rat Cytokine immunoassay (Bio-Rad Laboratories).
3
Protein Extraction from BeWo Cells
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5 × 106 BeWo cells were harvested, washed with PBS, and centrifuged at 1000× g for 10 min. The supernatant was carefully removed. Then, the precipitation was mixed and gently shaken with lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.5 mM PMSF, and 1 mM protease inhibitor cocktail. The suspended cells were lysed using ultrasonic sonicator (Qsonica, Newtown, CT, USA) on the ice and the 3-min-long lysis program was disrupting for 5 s at 50% amplitude followed by 10 s interval. Then, the lysis buffer was put on the ice for 40 min and cellular debris was removed by centrifugation at 15,000× g at 4 °C for 45 min. The resulting samples were treated immediately or stored at −80 °C until analysis. Protein concentration in the sample was measured by the modified Bradford method [27 (link)].
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