CytoScan™ SRB Cytotoxicity Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were washed twice with 1× PBS prior to fixing of cell.		- Absorbance was measured at 540 nm

Upstream tips
- Cells were washed twice with 1× PBS prior to fixing of cell.
Downstream tips
- Absorbance was measured at 540 nm

Publication protocol

Cells were plated into 96-well flat-bottom plates at a starting density of 104 cells per well. After an equilibration time of 12 to 24 h, until they reached 70%–80% confluency, culture media was removed and cells were treated in triplicate with increasing concentrations (1, 5, 10, 25, 50, 100, 200, 500, 1000, and 2000 µg Fe/mL) of the 10 different SPIONs suspended in culture media. Cell viability was then determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT assay; AMRESCO, Solon, OH, USA) and sulforhodamine B (SRB; CytoScanTM SRB Cytotoxicity Assay kit, G Biosciences, St. Louis, MO, USA) assays. The MTT assay determines the viability of cells according to their metabolic activity, whereas the SRB assay non specifically detects the total protein content in cells.
Briefly, the MTT assay was conducted by incubating cells with a 2 mg/mL solution of MTT (50 µL) for 4 h. The supernatant was then aspirated and 150 µL of dimethyl sulfoxide (DMSO) was applied to the cells to solubilize the formed formazan crystals. Plates were agitated and then centrifuged (3000 rpm for 10 min) to pellet the SPIONs. Finally, 100 µL of the supernatant was collected and transfered into a clean 96-well flat-bottom plate. Absorbance was measured at 540 nm using a microplate reader (SpectraMax i3 multi-mode platform, Molecular Devices, Sunnyvale, CA, USA).
The SRB assay was conducted in accordance to instructions provided with the commericial kit, except that cells were washed twice with 1× PBS prior to fixing of cell. Absorbance was measured at 540 nm using a microplate reader (SpectraMax i3 multi-mode platform, Molecular Devices).

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