Flow cytometry results, Figure 3, demonstrate that the initial population of MDA-MB-231 cells contain the sub-population of cells with the desired surface markers, CD44+/CD24-/low/CD45-. The flow cytometry data showed only ~4% of the total cells represent the sub-population with the desired surface markers. In a study using flow cytometry to identify stem-like cells, not including CD45 in their experimental analysis of MDA-MB-231 cells and similar cell types demonstrate that 90% of the population express the phenotype CD44+/CD24-/low phenotype (Fillmore & Kuperwasser, 2008). Other studies have found similar results looking at the same phenotype. In the current CD45 was added to the selection for stem-like cells and to flow cytometry assays.
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Interestingly, the differences between BCSC and the sorted control are more pronounced than the comparison with the depleted controls. These differences may be attributed to batch effect. Batch effect refers to the varying results in experiments often observed when experiments are performed at different times. Example causes often include changes in environment, such as temperature and humidity. This effect is most pronounced in RNA due to its lack of stability during common sequencing manipulations such as poly-A tail selection or reverse transcription polymerase chain reaction. To limit batch effect, procedures were performed in sequence until cDNA was produced and samples were frozen only when a stopping point was indicated in within the procedure. However, some groups of sequences were performed on separate days within the same fashion as previously described. Additional variability may be a result of varying times within ethanol based buffers during library preparation. When performing protocols with mulitple samples, a timer was used to limit variability of the effects of ethanol based solutions. The amount of time that ethonal is present on the samples determines the size of DNA or RNA reads that are attached to the beads; beads collect shorter lengths of DNA/RNA more readily and …show more content…
A significant difference in transcript expression is determined by a p-value of 0.05. The lack of differences among each condition could result from the sorting protocol used or the randomize sampling method for transcript selection during library preparation. As stated previously, sorting was performed using Magnetically-Activated Cell Sorting (MACS). Compared to Flourescence-Activated Cell Sorting (FACS), MACS based sorting have varying results between the two types of sorting (Herrid, Davey, Hutton, Colditz, & Hill, 2009). This is due to the amount of time required to perform MACS which can result in declines in cellular viability. To prevent cell death or decreases in viability due to being outside of growth medium, ice cold phosphate buffered saline (PBS) is used to preserve the cells. If the PBS is allowed to sit long enough, it can return to room temperature and become ineffective. Additonally, the depleted cells need to be removed from the columns while the magnetic beads are still attached to be collected. This removal can potentially tear open the cell membrane and kill cells surviving from the process. Finally, prior to freezing the cells, they must be allowed to relax in growth medium to allow the cells to chew remove any remaining magentic beads. Cell death can occur during this
Interestingly, the differences between BCSC and the sorted control are more pronounced than the comparison with the depleted controls. These differences may be attributed to batch effect. Batch effect refers to the varying results in experiments often observed when experiments are performed at different times. Example causes often include changes in environment, such as temperature and humidity. This effect is most pronounced in RNA due to its lack of stability during common sequencing manipulations such as poly-A tail selection or reverse transcription polymerase chain reaction. To limit batch effect, procedures were performed in sequence until cDNA was produced and samples were frozen only when a stopping point was indicated in within the procedure. However, some groups of sequences were performed on separate days within the same fashion as previously described. Additional variability may be a result of varying times within ethanol based buffers during library preparation. When performing protocols with mulitple samples, a timer was used to limit variability of the effects of ethanol based solutions. The amount of time that ethonal is present on the samples determines the size of DNA or RNA reads that are attached to the beads; beads collect shorter lengths of DNA/RNA more readily and …show more content…
A significant difference in transcript expression is determined by a p-value of 0.05. The lack of differences among each condition could result from the sorting protocol used or the randomize sampling method for transcript selection during library preparation. As stated previously, sorting was performed using Magnetically-Activated Cell Sorting (MACS). Compared to Flourescence-Activated Cell Sorting (FACS), MACS based sorting have varying results between the two types of sorting (Herrid, Davey, Hutton, Colditz, & Hill, 2009). This is due to the amount of time required to perform MACS which can result in declines in cellular viability. To prevent cell death or decreases in viability due to being outside of growth medium, ice cold phosphate buffered saline (PBS) is used to preserve the cells. If the PBS is allowed to sit long enough, it can return to room temperature and become ineffective. Additonally, the depleted cells need to be removed from the columns while the magnetic beads are still attached to be collected. This removal can potentially tear open the cell membrane and kill cells surviving from the process. Finally, prior to freezing the cells, they must be allowed to relax in growth medium to allow the cells to chew remove any remaining magentic beads. Cell death can occur during this