Background Info to experimental set-up:
Mutated Mucin-1 is found to be aberrantly expressed in diverse cancers and has been detected in certain mature B-cell malignancies such as Multiple Myeloma and Chronic Lymphocytic Leukemia.
A cell line of CLL was cultured and subsequently treated with GO-203 ( An experimental small molecule Inhibitor of Mucin-1 ).
There was a: 1. Non-Treatment control sample ( NT )
2. 5uM GO-203 Treated sample
3. 10uM GO-203 Treated sample
Samples each contained 5✖️10^5 cells. Incubated for 24 hours.
Samples were prepared for flow cytometry and stained with:
-APC conjugated Mucin-1 antibody ( To detect expression of Muc-1 ).
-PI to detect Necrosis and Late Apoptosis.
-Annexin V to detect Early apoptosis.
10,000 events were observed for each of the three samples.
The Query!
The expected result would be a decrease in Mucin-1 expression for the GO-203 treated samples.
However, upon data analysis of the MFI histogram charts for APC ( Detecting Muc-1 ), there is a single peak for the Non-Treatment and Double Peaks for the Treated samples.
In addition, the second peak of the Treated sample MFI graphs has a much higher fluorescence intensity than the first - Thus, Increasing the overall mean intensity level for the treated sample. Therefore the MFI value is higher in the GO-203 treated samples.
......Can someone explain what may be occurring?
.......Do I need to exclude all dead cells in my gating leaving only alive cells?
As in excluding PI-positive and FITC-positive cells??
Note
GO-203 causes apoptosis and ultimately cell death in the CLL cells. This was shown in PI/Annexin V graphs and subsequent ANOVA tests.
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