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: Triplicates of cells transfected with the same promoter were loaded at different positions of the plates (Figure ?) to ensure that the results do not change significantly over the time course of one flow cytometry measruement.

Figure 1: Time measurement 24 well. The bars indicate each triplicate loacted at a different position on the plate as shoan in the scheme below. The standard deviations of the replicates are indicated by the error bars.

Figure 2: Time measurement 96 well. The bars indicate each triplicate loacted at a different position on the plate as shoan in the scheme below. The standard deviations of the replicates are indicated by the error bars.

Table 1: 24 well loading scheme. The table reprents the wells of a 24 well plate and the pink colored boxes indicating the loading wells.

Table 2: 96 well loading scheme. The table reprents the wells of a 96 well plate and the pink colored boxes indicating the loading wells.

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, a standard measurement includes the constructs shown in table 1.

Sample negative control (transfection with a non-fluorescent plasmid p6) cotransfection with JeT plasmid and p6 cotransfection with JeT_CMV plasmid and p6 cotransfection with CMV plasmid and p6 cotransfection with mCherry reference plasmid and p6 cotransfection with CMV plasmid and mCherry reference plasmid cotransfection with JeT plasmid and mCherry reference plasmid cotransfection with JeT_CMV plasmid and mCherry reference plasmid

Figure 3: Flow cytometry results of the U2-OS Standard plate (1st measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the U2-OS cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above.The standard deviations of the replicates are also shown.

• We measured the 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, which includes the same samples as above (see table 1)

Figure 4: Flow cytometry results of the MCF-7 Standard plate (1st measurement).The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the MCF-7 cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above.The standard deviations of the replicates are also shown.

9-28-2009

• Second measurement of MCF-7 and U2-OS standard plates(same samples).

Figure 5: Flow cytometry results of the U2-OS standard plate(2nd measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the U2-OS cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above.The standard deviations of the replicates are also shown.

Figure 6: Flow cytometry results of the MCF-7 standard plate(2nd measurement).The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the MCF-7 cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above.The standard deviations of the replicates are also shown.

9-29-2009

• Third flow cytometry measurement of the MCF-7 and the U2-OS standard plates. Results are shown in the diagrams below.

Figure 7: Flow cytometry results of the U2-OS Standard plate (3rd measurement).The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the U2-OS cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above.The standard deviations of the replicates are also shown.

Figure 8: Flow cytometry results of the MCF-7 Standard plate (3rd measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the MCF-7 cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above. The standard deviations of the replicates are also shown.

9-30-2009

• Second measurement of the constitutive promoters in HeLa. Results shown below.

Figure 9: Flow cytometry results of the constitutive promoters in HeLa (1st measurement).The synthesized constitutive promoters of varying strength were measured 20 hours after transfection of the HeLa cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above.The standard deviations of the replicates are also shown .

10-02-2009

• We measured the NFkB responsive promoter plate in U2-OS 10 hours after induction.

Figure 10: Flow cytometry results of the NFkB responsive promoters in U2-OS (1st measurement). The .

10-03-2009

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

10-05-2009

• Three independent measurements of HeLa standard Plates, which include the same samples as above (see table 1)
• We measured the 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 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.

Figure 11: Flow cytometry results of the HeLa Standard plate (1st measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the HeLa cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above. The standard deviations of the replicates are also shown.

Figure 12: Flow cytometry results of the HeLa Standard plate (2nd measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the HeLa cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above. The standard deviations of the replicates are also shown.

Figure 13: Flow cytometry results of the HeLa Standard plate (3rd measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the HeLa cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above. The standard deviations of the replicates are also shown.

10-07-2009

• We measured the NFkB responsive promoter plate in U2-OS 10 hours after induction again.

Figure 14: Flow cytometry results of the NFkB responsive promoters in U2-OS (2nd measurement). The .

10-08-2009

• We measured the 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.
• We measured the SREBP responsive promoter in HeLa.
• Second measurement of the constitutive promoters in HeLa.

Figure 15: Flow cytometry results of the HeLa Standard plate (4th measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the HeLa cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above. The standard deviations of the replicates are also shown.

Figure 17: Flow cytometry results of the constitutive promoters in HeLa (2nd measurement).The synthesized constitutive promoters of varying strength were measured 20 hours after transfection of the HeLa cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above.The standard deviations of the replicates are also shown.

Figure 16: Flow cytometry results of the SREBP responsive promoters in HeLa (1st measurement). The .

10-11-2009

• We measured the natural promoter c-Jun (AP-1) in HeLa.

Figure 18: Flow cytometry results of the natural promoter c-Jun (AP-1). The .

10-12-2009

• Fifth measurement of the HeLa standard plate. Results shown below.
• Crossinduction of NF-kB responsive synthetic promoter NIIL10: We measured the promosing NKkB responsive promoter NIIL10 in different medium in U2-OS. Here, we want to see, if there is an induction of a nonspecific drug (Thiazolidin??) and how the different medium affect the promoter activity. No significant change.

Figure 19: Flow cytometry results of the HeLa Standard plate (5th measurement). The three standard promoters CMV, JeT, and JeT_CMV were measured 20 hours after transfection of the HeLa cells and their Relative Expression Units (REU) normalized to the JeT promoter are shown above. The standard deviations of the replicates are also shown.

Figure 20: Flow cytometry results of the NIIL10 promoter (NFkB responsive promoter) under different conditions in U2-OS. The .

10-13-2009

• We measured the 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: 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.

Figure 21: Time measurement 96 Well The .The standard deviations of the replicates are also shown.