For question 2, the purpose of taking confocal images is to see the binding affinity of free sgc8c and NR-sgc8c to CCRF-CEM cells. They found the NR-sgc8c (0.25nM) treated cells with very bright fluorescence confocal images , but no fluorscence was observed in the free sgc8c(0.25nM)treated cells. So that means NR-sgc8c has higher binding affinity to the receptos on the CCRF-CEM cell membrane.
1) Kd was determined with a non-linear fit equation. Y = Bmax * X / (Kd + X) where Y appears to be the fraction of fluorescence taken by subtracting the observed fluorescence (mean value of n=2-4) from the background and X appears to be the concentration in nM
2) Confocal microscopy was used to visualize the binding and fluorescence of the interacting antimers and cell proteins.
1) Kds were obtained by fitting the dependence of fluorescence intensity of specific binding on the concentration of the aptamers used to the following equation:
Y = BmaxX/(Kd + X).
2) Basically, by using a confocal pinhole, out of plane or scattered fluorescence is rejected. A very clean 3-D image is then able to be constructed from piecing together all the thin sections that are imaged from the fluorescence through the pinhole onto the very small area of your sample.
3) The aptamer is sgc8c. I do not know what the binding partner of the aptamer is.
1. To determine the Kd using flow cytometry, the authors plotted the concentration of their nanorod-DNA complex or fluorescein-tag labeled DNA versus the mean fluorescence obtained from the flow cytometry. This gave them a nonlinear plot (as shown in figure 4 of the paper), which they then fit to the equation of Y=BmaxX/(Kd+X). To determine the mean fluorescence they took there fluorescence obtained from their tag and subtracted the fluorescence obtained from using non-specific DNA. This makes sense because then the only fluorescence plotted would come the specific DNA-cell interaction.
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Comments
This paper describe the use of two techniques: flow cytometry and confocal microscopy.
1. How do they calculate the Kd from the flow cytometry data?
2. What was the purpose of using confocal microscopy?
3. What is the binding partner of the aptamer that is present in CCRF-CEM cells but not in Ramos cells?
Posted by: Edgar Arriaga | April 24, 2008 09:28 PM
For question 2, the purpose of taking confocal images is to see the binding affinity of free sgc8c and NR-sgc8c to CCRF-CEM cells. They found the NR-sgc8c (0.25nM) treated cells with very bright fluorescence confocal images , but no fluorscence was observed in the free sgc8c(0.25nM)treated cells. So that means NR-sgc8c has higher binding affinity to the receptos on the CCRF-CEM cell membrane.
Posted by: Yu-Shen Lin | April 26, 2008 02:26 PM
1) Kd was determined with a non-linear fit equation. Y = Bmax * X / (Kd + X) where Y appears to be the fraction of fluorescence taken by subtracting the observed fluorescence (mean value of n=2-4) from the background and X appears to be the concentration in nM
2) Confocal microscopy was used to visualize the binding and fluorescence of the interacting antimers and cell proteins.
Posted by: Chad Satori | April 26, 2008 07:43 PM
1) Kds were obtained by fitting the dependence of fluorescence intensity of specific binding on the concentration of the aptamers used to the following equation:
Y = BmaxX/(Kd + X).
2) Basically, by using a confocal pinhole, out of plane or scattered fluorescence is rejected. A very clean 3-D image is then able to be constructed from piecing together all the thin sections that are imaged from the fluorescence through the pinhole onto the very small area of your sample.
3) The aptamer is sgc8c. I do not know what the binding partner of the aptamer is.
Posted by: Courtney Wettlaufer | April 27, 2008 09:41 PM
1. To determine the Kd using flow cytometry, the authors plotted the concentration of their nanorod-DNA complex or fluorescein-tag labeled DNA versus the mean fluorescence obtained from the flow cytometry. This gave them a nonlinear plot (as shown in figure 4 of the paper), which they then fit to the equation of Y=BmaxX/(Kd+X). To determine the mean fluorescence they took there fluorescence obtained from their tag and subtracted the fluorescence obtained from using non-specific DNA. This makes sense because then the only fluorescence plotted would come the specific DNA-cell interaction.
Posted by: Jonathan Dozier | April 27, 2008 11:31 PM