HL-60 cells are immortal cells, and were derived from a patient with acute promyelocytic leukemia (Collins, 1987). HL- 60 cells require simple maintenance in vitro, and they are multipotent, which means that they are able to form multiple cell types, but are restricted to a specific lineage (Cirtain et al., 2016). HL-60 cell are invaluable as they are useful in science, easy to maintain, easy to differentiate. The HL-60 cell line are useful for studying cellular and molecular mechanisms involved in the proliferation and differentiation of normal and leukemic cells of the hematopoietic, or white blood cell lineage (Collins, 1987). Various chemical agents such as PMA and DMSO have been used to induce differentiation via signal transduction in
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2016. BIOL 302 Cell and Molecular Biology Laboratory Manual. University of South Carolina, Columbia, SC. For the experiment, we used β-actin as a positive control. β-actin is a highly distributed protein in many cell types and, because of this, it is great to use as a positive control when analyzing gene expression (Cirtain et al., 2016). We also use many replications in the experiment for accurate results. We used four PMA treatment and four DMSO treatments, and treatment was given both primers. Results:
After performing the PCR, we found that DMSO treatment mostly produced β-actin while most PMA treatment produced both MMP-9 and β-actin. But lane 2 for DMSO treatment shows light band, which means it produced MMP-9 primer; as DMSO does produce some monocytes. In the same way, lane 6 and 7 for PMA treatment did not show any primers (Figure1).
Figure 1: The results of RT-PCR showing gene expression of MMP-9 and β-actin in DMSO and PMA treatment of HL-60 Cells. The upper bands show MMP-9 480bp, while the lower bands are β-actin 314bp. The plus (+) sign means presence of that primer while negative (-) means absent of that primer. …show more content…
We concluded that DMSO treated HL-60 cells produce mainly β-actin primers, while PMA treated produced both MMP-9 and β-actin. These data supports our hypothesis and refutes the null hypothesis. But as it can be seen in figure 1, lane 3 for DMSO also amplified MMP-9. The reason behind that result is, although DMSO produces mostly granulocytes, it can also produce monocytes. And monocytes stimulate the regulation of MMP-9, so lane 3 shows spontaneous differentiation. For lane 6 in PMA treatment, there was no β-actin. This indicted something went wrong in the experiment and RNA was not successfully isolated from the treated HL-60 cells. In PMA treatment, lane 7 did not amplify MMP-9 primer. As the cell viability is much lower in PMA than in DMSO, it may have an effect the results. As you would expect to have less cells to work with for the PMA treatment than the DMSO treatment. For all other lanes, RNA was isolation was successful, as it has the presence of β-actin. Although this is the result we expected based on previous research, we also found that DMSO could amplify MMP-9. Our results are not limited, as we did not just use one-person data. We had a large sample size data, as we used the data of the whole class. But additional replications could be helpful, as larger data would provide more accurate result. Some of the errors in the experiment could be not following
2016. BIOL 302 Cell and Molecular Biology Laboratory Manual. University of South Carolina, Columbia, SC. For the experiment, we used β-actin as a positive control. β-actin is a highly distributed protein in many cell types and, because of this, it is great to use as a positive control when analyzing gene expression (Cirtain et al., 2016). We also use many replications in the experiment for accurate results. We used four PMA treatment and four DMSO treatments, and treatment was given both primers. Results:
After performing the PCR, we found that DMSO treatment mostly produced β-actin while most PMA treatment produced both MMP-9 and β-actin. But lane 2 for DMSO treatment shows light band, which means it produced MMP-9 primer; as DMSO does produce some monocytes. In the same way, lane 6 and 7 for PMA treatment did not show any primers (Figure1).
Figure 1: The results of RT-PCR showing gene expression of MMP-9 and β-actin in DMSO and PMA treatment of HL-60 Cells. The upper bands show MMP-9 480bp, while the lower bands are β-actin 314bp. The plus (+) sign means presence of that primer while negative (-) means absent of that primer. …show more content…
We concluded that DMSO treated HL-60 cells produce mainly β-actin primers, while PMA treated produced both MMP-9 and β-actin. These data supports our hypothesis and refutes the null hypothesis. But as it can be seen in figure 1, lane 3 for DMSO also amplified MMP-9. The reason behind that result is, although DMSO produces mostly granulocytes, it can also produce monocytes. And monocytes stimulate the regulation of MMP-9, so lane 3 shows spontaneous differentiation. For lane 6 in PMA treatment, there was no β-actin. This indicted something went wrong in the experiment and RNA was not successfully isolated from the treated HL-60 cells. In PMA treatment, lane 7 did not amplify MMP-9 primer. As the cell viability is much lower in PMA than in DMSO, it may have an effect the results. As you would expect to have less cells to work with for the PMA treatment than the DMSO treatment. For all other lanes, RNA was isolation was successful, as it has the presence of β-actin. Although this is the result we expected based on previous research, we also found that DMSO could amplify MMP-9. Our results are not limited, as we did not just use one-person data. We had a large sample size data, as we used the data of the whole class. But additional replications could be helpful, as larger data would provide more accurate result. Some of the errors in the experiment could be not following