Nitro Blue Tetrazolium (NBT) Reduction Assay

Differentiation of HL-60 cells was assayed by NBT reduction test. The cells were treated by A. Urmiana extracts and incubated 96 hr. Then the cells were collected and washed 2 times in culture medium and resuspended in the same medium containing 20% FCS. 100µl of cells suspension (2 × 106 cells/ml) were transferred to 96 micro-wells. Phorbol 13 Myristate Acetate (PMA) (5ng/ml) (Sigma, U.S.A.) was used as positive control stimulator for NBT reduction by cells. The cells were incubated in the presence of freshly prepared NBT solution (1 mg/ml in PBS) for 45 min in a CO2 incubator. Then the cells were washed 3 times with PBS and the percentage of the cells that stained dark blue-black with formazan deposits were determined under a light microscope using a glass slide. Minimum 300 numbers of the cells were scored from each treatment.

Because of low NBT reduction results in using A. urmiana extracts, the cells were treated with different concentrations of Artemia extracts separately and/or in combination by Retinoic Acid (RA)(1µg/ml) too. NBT assayed was done as described above in combination study.