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

⇐ Main Page

⇐ 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

1/10/2010

seed two 96-well plates with HEK cells 5000 cells/well

2/10/2010

transfection

11/10/2010

• transfection of 24-well plates for 50 clones of shuffled capsid
• coat 6-well plates with collagen for primary hepatocytes
• measurements of miMeasure

12/10/2010

• seed 1 96-well plate for transfection with miMeasure
  • 6 lines Huh7
  • 6 lines HepG2
• Dual Luciferase assay for ??
• seed 24-well plates with Huh7 for second selection round

13/10/2010

• seeding of primary hepatocytes for first selection round of capsid library on a 6-well plate
• seeding of primary hepatocytes for selection round of 50 clones on 2 24-well plates

14/10/2010

• transfection of 1 96-well plate with off targeting constructs:
  • M23-M29 with or without co-transfection of miR122 of miRsag as a control
• transfection of tuning construct K4 with or without co-transfection of miRsAg and miRhaat as a control

15/10/2010

• Dual luciferase assay on tuning construct with co-transfection of shRNA miRsag and miR122
• Dual luciferase assay of off target constructs
• seeding of :
  • 1 96-well plate of half Huh7 and half HepG2
  • 1 plate for testing nuclei staining with Hela, HepG2 and Huh7
• prepare 3 bottles of media
• organize 500 Million Hek293T cells :)
• change media from primary hepatocytes

15/10/2010

• plate 1 96-well plate with half of the wells with Huh7 and other half with HepG2
• plate half of a 96-well plate with Hela, HepG2 and Huh7 for testing nuclei staining for TECAN

16/10/2010

• transfect 96-well plate of liver cell lines with miMeasure with perfect, imperfect 9-12 and imperfect 9-22 binding site
• transfect test staining plate with miMeasure in different concentrations:
  • 10ng of miMeasure in 3 wells of each cell line
  • 30ng of miMeasure in 3 wells of each cell line
• plate 8 96-well plates with Hela cells
• plate 1 96-well plate with half Huh7 and HepG2

17/10/2010

• transfect 3 96-well plates with tuning construct for in vivo:
  • tuning construct M1-M10 without any synthetic miRNA
  • co-transfection of tuning construct M1-M10 with synthetic miRhaat
  • co-transfection of tuning construct M1-M10 with synthetic miRSag
• transfect 3 96-well plates with miMeasure with different binding sites against miRsag:
  • miMeasure M12-M22 without any synthetic miRNA
  • co-transfection of miMeasure M12-M22 with synthetic miRhaat
  • co-transfection of miMeasure M12-M22 with synthetic miRSag
• coat 1 6-well plate with collagene for primary hepatocytes

18/10/2010

• measure 3 96-well plates with tuning construct for in vivo by Dual luciferase assay
  • measurement did not work, no luciferase activity could be measured
• measure 1 96-well plate of miMeasure with binding sites for miR122
  • measurement did not work, maybe too little DNA transfected in the cells (10ng) as Huh7 are not easily transfected
• measure plate for testing nuclei staining
  • measurement did not work
• coat plates with collagene (2* 6-well plate) for primary hepatocytes
• plate 1 96-well plate with Huh7 and HepG2
• plate 24 well plate with Hela cells for haat Elisa

19/10/2010

• transfect miMeasure into Huh7 and HepG2 on 96-well plate again with binding sites of miR122:
  • perfect binding site
  • imperfect binding sites (randomized 9-12)
  • imperfect binding sites (randomized 9-22)
• transfect haat cDNA constructs into Hela cells on 24-well plates

• measurement M12-M19
  • TECAN
  • did not work, because cells were clustered
  • Confocal
  • cells from the 96 well plate were pooled together and single pictures were made.

File:M12-M22 confocalS.pdf

• who deleted what I wrote here??? hallooo?? hmm I guess I just didn't save it. damn.

23/10/2010

• measurement of Hela and Hek Cells transfected with miMeasure M12-M22. Four conditions were transfected with four replicates each: a = not cotransfected, b = cotransfected with miRsAg on pcDNA5, c = cotransfected with empty pcDNA5 (CMV promoter), d = cotransfected with pcDNA5 containing different shRNA (shRNA3 from Elena).
  • because of a contamination, not all replicates could be considered. Plates were screened for positive transfections
  • cells were trypsinated (30µl Trypsin) for 10min and then resuspended with 170µl PBS/BSA. Replicates for each condition were pooled into 24 well plates.
  • 200µl of each was transferred to a 96 well plate for FACS measurement.
  • 100µl of each was used for confocal analysis