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References and notes
13. Cell viability assay: Novel synthetic compounds 3a–g were first evaluated for its
antiproliferative activity in U937 and K562 cells using MTS–PMS assay. Briefly,
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U937 and K562 cells (5 ꢀ 104/200
l
l) were seeded in 96 well tissue culture
M) in triplicate for 48 h at
plates and incubated with the compounds (0–100
l
37 °C, 5% CO2. To evaluate cell viability, MTS [3-(4,5 dimethylthiazol-2-yl) 5-
(3-carboxymethoxy-phenyl)-2-(4-sulphonyl)-2H-tetrazolium, inner salt]
(2.0 mg/ml) and PMS (Phenazine methosulfate) (0.92 mg/ml) were added in
a ratio of 10:1 (20 l per well) and plates incubated for 3 h at 37 °C. Resultant
l
absorbance was measured at 490 nm in an ELISA reader. The specific
absorbance that represented formazan production was calculated by
substraction of background absorbance from total absorbance. The mean %
viability was calculated as follows:
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Mean specific absorbance of treated cells
ꢀ 100
Mean specific absorbance of untreated cells
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15. Flow cytometric analysis of externalized phosphatidylserine: U937 cells were
incubated with an IC50 dose of 3g (13.06 lM) for 8 h at 37 °C, 5% CO2. Cells
were centrifuged (4000 rpm for 4 min), washed twice in phosphate buffered
saline (0.02 M, pH 7.2, PBS) and re-suspended in Annexin V binding buffer
(10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Annexin V-FITC and
propidium iodide were then added and incubated for 15 min in dark at 25 °C
according to the manufacturer’s instructions. Data acquisition was done on a
FACS Calibur flow cytometer (BD Biosciences, USA) and analyzed with CELL QUEST
PRO software.
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