Team:Heidelberg/Notebook measure/NotebookFC

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=Notebook Flow Cytometry=

9-21-2009

 * All cells for flow cytometry were transfected with the promoter of interest in front of GFP and the reference plasmid with JeT and mCherry in a 2:1 ratio. This ratio was chosen to ensure that we have GFP in every cell expressing mCherry and thereby increasing the number of double positive cells.
 * Time course measurement of 24 and 96 well plate (Fig. 1-2): Triplicates of cells transfected with the same promoter were loaded at different positions of the plates (Fig. 3-4) to ensure that the results do not change significantly over the time course of one flow cytometry measurement.

9-24-2009

 * We performed standard measurements in HeLa, U2OS and MCF7 with our three standard promoters: CMV, JeT and JeT-CMV. These measurements were performed in 3 independent measurements. The initial results of U2OS standard are shown below (Fig. 3), a standard measurement includes the constructs shown in table 3.


 * We measured the synthetic constitutive promoters in the HeLa cell line. Unfortunately, the number of the viable cells are not enough for a reliable measurement.

9-25-2009

 * Flow cytometric measurement of MCF-7 standard plate (Fig. 4), which includes the same samples as above (see table 3).

9-28-2009

 * Second measurement of MCF-7 (Fig. 6) and U2-OS (Fig. 5) standard plates(same samples).

9-29-2009

 * Third flow cytometry measurement of the MCF-7 (Fig. 8) and the U2-OS (Fig. 7) standard plates. Results are shown in the diagrams below.

9-30-2009

 * Second measurement of the synthetic constitutive promoters in HeLa. Results shown below (Fig. 9).



10-02-2009

 * We measured the synthetic NFkB responsive promoter plate in U2-OS after 10 hours of induction with TNF-alpha (Fig. 10).



10-03-2009

 * We measured the synthetic HIF responsive promoter in MCF-7. Unfortunately, the cell number was too low.

10-05-2009

 * Three independent measurements of HeLa standard plates (Fig. 11-13), which include the same samples as above (see table 3)
 * We measured the synthetic constitutive promoters in the HeLa cell line again. Unfortunately, the number of the viable cells are not enough for a reliable measurement.
 * We measured the synthetic AhR responsive promoters and the natural promoter CYP1A1 (AhR responsive promoter) in HeLa. Unfortunately, the number of the viable cells are not enough for a reliable measurement.

10-07-2009

 * We measured the synthetic NFkB responsive promoter plate in U2-OS after 10 hours of induction again (Fig. 14).



10-08-2009

 * We measured the synthetic Estrogen responsive promoter in MCF-7. Unfortunately, the number of the viable cells are not enough for a reliable measurement.

10-09-2009

 * Fourth measurement of the HeLa Standard plate (Fig. 15).
 * We measured the synthetic SREBP responsive promoter in HeLa (induction with LDS). -> no successfull induction (Fig. 17).
 * Second measurement of the synthetic constitutive promoters in HeLa (Fig. 16).

10-11-2009

 * We measured the natural promoter c-Jun (AP-1) in HeLa after induction with Epidermal Growth Factor (Fig. 18).



10-12-2009

 * Fifth measurement of the HeLa standard plate. Results shown below (Fig. 19).
 * Crossinduction of NF-kB responsive synthetic promoter NIIL10 (Fig. 20): We measured the promosing NFkB responsive promoter NIIL10 (clone 31) in different media in U2-OS. Here, we want to see, if there is an induction of a nonspecific drug (Thiazolidinedione, 1:100 000) and how the different media affect the promoter activity. No significant change -> no influence/induction of the drug or medium.

10-13-2009

 * We measured the synthetic p53 responsive promoter in MCF-7. Unfortunately, there were some problems with the flow cytometry analysis.

10-14-2009

 * We measured the standard CMV promoter under different conditions (Fig. 21): full medium, minimal medium, and minimal medium + Erolimus. We wanted to check that CMV is comparable under different conditions. A fourth sample prepared at the same time will be measured after 50 hours to analyze the change of JeT and CMV over a long period of time.
 * Results of the first three measurements shown below.



10-15-2009

 * We measured the MCF-7 (Fig. 22) and U2-OS (Fig. 23) Standard plate (fourth measurements).


 * We measured a second timepoint (after 50 hours) of the CMV promoter and added these results in the figure 21 (new figure 24, below). To check, if the GFP fluorescence changed after 50 hours.
 * Third measurement of NIIL10 (NFkb responsive) in U2-OS (Fig. 25).