Viable cells were counted using trypan blue staining and a haemocytometer. 2500 cells (5000 BxPC-3 cells) were seeded onto clear 96-well plates (Corning). Wells containing only media and vehicle acted as blanks. Plates were then incubated for 2–96 h. At each time-point, 10 μL of CCK-8 (Dojindo, Munich, Germany) was added to each well and plates were incubated for another 60 min at 37 °C. Following incubation, the plate was read for absorbance at 450 nm using a Synergy HT plate reader, operated by Gen 5 software (both Biotek, Whiting, Vermont, USA). Absorbance from blank wells containing only DMEM and vehicle were subtracted from each well reading. Cells were then fixed using of 100 μL 10% trichloroacetic acid at 4 °C for 1 h, washed with milliQ water and dried at 50 °C. Fixed cells were stained using 100 μL 0.057% sulforhodamine-B (SRB) (Sigma) dissolved in 1% acetic acid. Unbound dye was washed off of the cells using 1% acetic acid and plates were then dried at 50 °C. Bound dye was re-suspended in 200-μL 10-mM Tris-Base solution (Sigma) and absorbance at 540 nm was read using the Synergy HT plate reader. There were 4 replicates for each condition in every experiment.
Synergy ht
Manufactured by Merck Group
Synergy HT is a multi-mode microplate reader designed for a variety of applications in life science research and drug discovery. It features high performance and flexibility, allowing for the detection of multiple detection modes including absorbance, fluorescence, and luminescence.
Automatically generated - may contain errors
Lab products found in correlation
Tris base solution, by Merck Group (1 mentions)
Clear 96 well plate, by Corning (1 mentions)
Sulforhodamine b srb, by Merck Group (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Resorufin, by Merck Group (1 mentions)
Resazurin, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
3 protocols using synergy ht
1
Cell Viability Assay with CCK-8 and SRB
2
Antimycobacterial Activity Assay of C10
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Logarithmically growing Mtb was inoculated into Sauton’s medium in 96 well plates with wells containing increasing concentrations of C10. M. tuberculosis was inoculated at an ODλ600 of 0.0025 in 200 μL per well. The plates were incubated at 37°C in 5% CO2 for 1 week, at which point 32.5 μL of a mixture containing an 8:5 ratio of 0.6 mM resazurin (Sigma) dissolved in 1X phosphate-buffered saline to 20% Tween 80 was added, and the production of fluorescent resorufin was measured on a Synergy HT plate reader with excitation λex = 530 nm and emission λem = 590 nm after incubation at 37°C in 5% CO2 overnight. For each assay, medium alone served as a negative control, and untreated Mtb was included as a positive control. The percent inhibition was calculated as the {[(fluorescence of the positive control – fluorescence of the negative control) – (fluorescence of the sample – fluorescence of the negative control)]/(fluorescence of the positive control – fluorescence of the negative control)} × 100.
3
MTT Assay for NP Cytotoxicity
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MTT assay was used to determine cell metabolic activity after NPs incubation in the dark, irradiation, or both after 24 h. This colorimetric assay is measuring the ability of bladder cancer cells to reduce yellow 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma), to a purple formazan on a microplate reader (Synergy HT). The results are expressed in percentage of control (i.e. optical density of formazan from cells not exposed to NPs).
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