From 2009.igem.org

Revision as of 15:37, 15 October 2009 by Chiller (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Notebook Flow Cytometry

9-21-2009

We have the GFP:Cherry ratio 2:1 to ensure that we have GFP in every cell expressing mCherry and thereby increasing the number of mCherry cells that also contain GFP.
Time course measurement of 24 and 96 well plate. Triplicates of the same transfection reaction were loaded at different positions in the plate to check wether the results change during the time course of the experiment.

Figure ?: Time measurement 24 Well The .The standard deviations of the replicates are also shown.

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

9-24-2009

We performed standard measurements in HeLa, U2OS and MCF7 with our three standard promoters: CMV, JeT and JeT-CMV. These measurements will be performed in 3 independent measurements. The initial result of U2OS is shown below, which includes the following samples (see 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 ?: 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

We measured the MCF-7 Standard plate, which includes the same samples as above (see table 1)

Figure ?: 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

We measured the MCF-7 and the U2-OS Standard plate again (same samples).

Figure ?: 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 ?: 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

We measured the MCF-7 and the U2-OS Standard plate the third time (same samples).

Figure ?: 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 ?: 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

We measured the constitutive promoters in the HeLa cell line again.

Figure ?: 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 ?: 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

We measured the HeLa Standard plate in three different experiments????, which includes 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 ?: 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 ?: 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 ?: 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 ?: 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

We measured the HeLa Standard plate (fourth time).
We measured the SREBP responsive promoter in HeLa.
We measured the constitutive promoters in HeLa again.

Figure ?: 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.

File:.png

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

Figure ?: 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.

10-11-2009

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

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

10-12-2009

We measured the HeLa Standard plate (fifth time).
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 ?: 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 ?: 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.