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The Use of HL-60 Cell line to Study Cell Differentiation and Examine the Cellular Functions; Phagocytosis and Exocytosis using PMA and DMSO.
Materials and methods:
Live / dead staining:
HL-60 cell line culture was incubated for a week, and a culture of HL-60 culture with 1 million cells/mL concentration was used as control. HL-60 cell line control culture was added to 500 ÂµL of sterile PBS. Then 1ml of control culture was added to first row of the 24 well plate and a serial of dilutions were prepared moving across the plate from left to right except the last column which was used as a background for the fluorescence. Under low light condition, 10% triton x was added to the standard culture to be stained with 51 ÂµL PI and calcein “AM to reach the final concentration of 0.2%. Then it was incubated at 37ËšC for 20 minutes. After that it was examined using fluorescence microscope, and then both standard and sample plates were examined using fluorescent plate reader.
HL-60 cells differentiation with PMA and DMSO:
Two 24 well plates of HL-60 culture were prepared. PMA was used to treat one plate to induce differentiation to macrophage-like cells while DMSO was used to treat the other plate to induce the differentiation to granulocyte-like cells. Then the calculated volumes to provide 100,000 cells were added to PMA plate and to DMSO plate. 5% FCS in DMEM was added into the two plates to reach 500 ÂµL total volumes in each well. The two prepared plates were incubated at 37ËšC for 3 days. Then PMA plate was analysed using live and dead staining and inverted microscope to determine the best concentrations of PMA for the differentiation.