Measurement Standard

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⇐ Measurements

08/08/2010

seeding cells for test measurements - 96 well plate for plate-reader and FACS

• cells were grown in DMEM 10%FBS with phenol red
• washed with PBS
• trypsinised (2 ml trypsin)
• 5ml of OptiMEM media (no FBS) added
• counted cells: HeLa 3.8*10^6 cells/ml, HEK 3.3*10^6 cells/ml, HEK T-Rex 1.5*10^6 cells/ml, Huh7 0.8*10^6 cells/ml
• seeded 5000 and 2500 cells/well as on the scheme 96well plate 080810.jpg
• media: DMEM +L-Glu +PenStrep +10%FBS, OptiMEM +PenStrep, OptiMEM +PenStrep +2%FBS
• in all wells where T-Rex cells were seeded zeocin and blasticin were added, in wells with Huh7 cells - non essential amino acids

-> cells did not adhere well, coat plate with poly-L-lysine and repeat

09/08/2010

seeding and transfecting cells for microscopy test measurements

• add 0.6ul FuGENE reagent to 20ul of OptiMEM
• mix and incubate 5' at RT
• add 0.2ug DNA
• mix and incubate 15' at RT
• add DNA-FuGENE solution to 10 000 cells (400ul)
• mix and incubate 10' 37degC 300rpm (shaker for 15ml falcon tubes is in the cell culture room at 3rd floor)
• transfere cells to the plate
• grow 48h

10/08/2010

coating plates with poly-L-lysine

• add 15ul of poly-L-lysine solution to each well (make sure whole surface is covered with solution)
• leave for 30' in the incubator
• remove poly-L-lysine solution
• wash once with PBS

new 96-well plate was prepared (wells 2500 cells Huh7 are contaminated with HeLa) like previously

25/08/2010

seeding cells for pilot FACS and Tecan measurements (96-well plate)

26/08/2010

transfection - 96-well plate

27/08/2010

pilot FACS and Tecan measurements (96-well plate)

⇓ September

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⇐ Measurements

⇑ August

02/09/2010

• seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
  • purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression
  • problem: forgot to coat the plate, therefore cells did not attach

03/09/2010

• transfection of cells for fluorescent measurement (EGFP and EBFP2):

• set-up of the plate:
  • non-transfected cell line
  • each cell line transfected with EGFP control plasmid
  • each cell line transfected with EBFP2 control plasmid
  • each cell line double-transfected with EGFP and EBFP2 control plasmids
  • Results:
    • too little cells have been plated
    • Huh7 cells are hard to transfect
    • expression of EBFP2 is much lower than expression of EGFP

04/09/2010

• seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
  • purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression

05/09/2010

• transfection of cells for fluorescent measurement (EGFP and EBFP2):
• set-up of the plate:
  • non-transfected cell line
  • each cell line transfected with EGFP control plasmid
  • each cell line transfected with EBFP2 control plasmid
  • each cell line double-transfected with EGFP and EBFP2 control plasmids

06/09/2010

• TECAN and FACS pilot measurements of cells transfected with EGFP and EBFP2

07/09/2010

• seeding and transfection of HeLa and HEK cells for pilot microscopy measurements:

09/09/2010

• pilot microscopy measurements:

20/09/2010

• seeding 2 96-well plates with 5000 cells each well:
  • test whether tuning construct is properly expressing firefly and renilla luciferase and thereby testing whether every part is functional

21/09/2010

• transfection of both 96-well plates with K1-K8:
  • transfect 50ng of each construct
  • check for promoter strength and functionality of the constructs

22/09/2010

• dual luciferase assay on the 2 96-well plates:
  • all constructs are functional as firefly and renilla luciferase are expressed
  • transfection of construct into Hek t-Rex cells reveals that the CMVTetO2 (construct K4) is also functional as the expression of firefly to renilla counts is nearly zero

23/09/2010

dual luciferase assay