Team:Heidelberg/Project/miMeasure
From 2010.igem.org
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First, the cells were washed with 1x PBS and detached from the plate using Trypsin. 30µl Trypsin was added to each well, incubated for ten minutes at room temperature. Cells were resuspended in 170µl 1%BSA in PBS and replicates for each condition were pooled into 24 well plates. 200µl from each well were used for FACS measurements, 100-150µl were used for confocal microscopy. | First, the cells were washed with 1x PBS and detached from the plate using Trypsin. 30µl Trypsin was added to each well, incubated for ten minutes at room temperature. Cells were resuspended in 170µl 1%BSA in PBS and replicates for each condition were pooled into 24 well plates. 200µl from each well were used for FACS measurements, 100-150µl were used for confocal microscopy. | ||
- | FACS | + | ===FACS=== |
- | Microscopy | + | ===Microscopy=== |
Single images were obtained using the Leica TCS SP5 confocal microscope and ??? camera with the Leica AF6000 imaging software ??. GFP fluorescence was excited by Argon 488nm laser and measured at xx-xxnm, BFP fluorescence was excited by UV laser at 405nm and measured at xx-xxnm. Pictures were taken sequentially line by line in three different channels for GFP, BFP and bright field. | Single images were obtained using the Leica TCS SP5 confocal microscope and ??? camera with the Leica AF6000 imaging software ??. GFP fluorescence was excited by Argon 488nm laser and measured at xx-xxnm, BFP fluorescence was excited by UV laser at 405nm and measured at xx-xxnm. Pictures were taken sequentially line by line in three different channels for GFP, BFP and bright field. | ||
- | Data Analysis | + | ===Data Analysis=== |
To analyze the fluorescence of single cells, we segmented the images using ImageJ. In 8bit pictures, we set the threshold for each channel to 50, thereby filtering the background. This allows us to annotate cells automatically using the “analyze particles” tool. We could now get the fluorescence intensity for each single cell on each channel (GFP or BFP) as an 8bit output, i.e. a value between 50 and 255. Form this data, we calculated the GFP:BFP ratio. We could then visualize the mean of these rations in a bar plot or use all the data to calculate a linear regression curve. | To analyze the fluorescence of single cells, we segmented the images using ImageJ. In 8bit pictures, we set the threshold for each channel to 50, thereby filtering the background. This allows us to annotate cells automatically using the “analyze particles” tool. We could now get the fluorescence intensity for each single cell on each channel (GFP or BFP) as an 8bit output, i.e. a value between 50 and 255. Form this data, we calculated the GFP:BFP ratio. We could then visualize the mean of these rations in a bar plot or use all the data to calculate a linear regression curve. |
Revision as of 10:54, 24 October 2010
![](https://static.igem.org/mediawiki/2010/9/92/Bg_plasmid.png)
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