July 2:

• HepG2 cell passaging
• Made sodium palmitate from powder to solution by 50% Ethanol

July 3:

• Used GCMS to quantify the FA in the medium

July 4:

• Passaged HepG2 cells
• Changed medium for cell line
• Searched for information of MTT assay and Transfection by lipofectamine
• Got GCMS results for the last day: Not accurate/usable

July 5:

• Repeated GCMS test again testing FA in medium and we made
• Got the result of GCMS: Not accurate/usable

July 8:

• Passaged HepG2 cells
• Used some HepG2 cells to extract genomic DNA

July 9:

• Another try of GCMS with a gradient concentration of FA
• Got the result of GCMS: Not match with the concentration we made

July 10:

• Repeated GCMS: No accurate results
• Changed medium for the cell line
• Checked lipofectamine protocol

July 11:

• GCMS again for the same sodium palmitate we made: No accurate results
• Passaged HepG2 cell line

July 12:

• Prepared reagents for MTT assay for cell viability test, protocols checked
• Changed the medium of the cell line

July 15:

• Seeded HepG2 cells into 96-well plate for MTT cell viability test and adding FA
• Seeded HepG2 cells into 96-well plate for transfection of PEGFP-N1

July 16:

• Transfection of PEGFP-N1 into HepG2 cells
• Passaged HepG2 cells

July 17:

• Changed the medium after transfection had been done in 24 hours
• Did MTT assay and getting result (adding FA in the wrong time, cannot be used)
• Made new DMEM
• Changed medium for the cell line

July 18:

• Seeded HepG2 cells in 24-well plate for transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Got polylysine for coating

July 19:

• Changed the medium for the HepG2 cell line
• Seeded cells for another MTT assay
• Checked HepG2 cells for transfection: growing slow, 30% confluency, cannot do transfection
• Seeded cells on a 96-well plate for transfection in the coming week
• Passaged HepG2 cells

July 22:

• Transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP on 96-well plate
• Added gradient concentration of sodium palmitate into HepG2 cells for MTT assay
• Coated coverslips with polylysine for HepG2 cell staining and fixation

July 23:

• Replaced the medium after transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Finished MTT assay to get cell viability in different FA concentrations in 24 hours
• Transferred cells containing PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into 24-well plate with well-coated coverslips in each well

July 24:

• Stained mitochondria by Mito-tracker@Red and fixating by formaldehyde of HepG2 cells transfected with pEGFP-N1, pCMV/myc/mito/EGFP and Pmv/myc/mito/GFP in 24-well plate
• Passaged HepG2 cells and changed medium of the cell line

July 25:

• Got results for cell staining and fixation: No obvious overlapping pattern between GPF signal and Mito-tracker red.

July 26:

• Coated coverslips by polylysine
• Seeded HepG2 cells in one 24-well plate for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Seeded HepG2 cells on two 96-well plate, for transfection of FABP construct and MTT assay in 48 hours

July 29:

• Transfection of FABP construct with pEGFP-N1 as control
• Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24-well plate with well-coated coverslips
• Added gradient concentration form 0.1mM to 1.2mM of FA into the plate for MTT assay

July 30:

• Changed medium after transfection of pEGFP-N1 and FABP construct and saw the results: Both pEGFP-N1 and FABP construct did not show GFP signals
• Changed medium after transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Did staining and fixation of the coverslips and saw the results: No obvious overlapping patterns of GFP signals and Mito-tracker red
• Passaged HepG2 cells

Aug 1:

• Seeded cells for transfection of pEGFP-N1, FABP construct, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Changed medium for HepG2 cell line
• Got results for MTT assay in 48 hours

Aug 2:

• Checked if transfection (after 22 hrs) worked
• No GFP signal for pEFGP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP

 

Aug 5:

• Transfection of HepG2 cells on 10cm Plates
• Passaged P(n+8) HepG2 cells into one P(n+9)
• Seeded HepG2 cells on 96-well plates for transfection

Aug 6:

• Trouble shooting for transfection of HepG2 cells on 96 well plates

Aug 7:

• Observe transfected cell. GFP signal expressed
• Seeded cells for transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well-plate
• Seeded cells for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24 well plate with polylysine coated coverslips
• Seeded cells for MTT assay

Aug 8:

• Observed transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well-plate. No GFP signal observed
• Observed transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP. GFP signal observed
• Stained mitochondria of cells transfected with pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP using mito tracking dye
• Observed cell with both transfection and stained mitochondria: No GFP signal observed because transfection occurred with very low efficiency.
• Prepared new cell line: HEK293FT
• Conducted MTT assay

Aug 12:

• MTT Assay without seeding and incubation
• Changed medium for HepG2 and HEK293FT cell lines
• Prepared polylysine coated coverslips

Aug 13:

• Seeded cells using HEK293FT cell line for transfection

Aug 15:

• Cells detached and not in good condition for transfection

Aug 16:

• Seeded cells HEK293FT cell line for transfection

Aug 19:

• Transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well plate and pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24 well plate.

Aug 20:

• Seeing results of transfection of FABP construct and pEGFP-N1 in HEK293FT cells and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals

Aug 22:

• Transfection of pEGFP-N1 and FABP construct into HEK293FT cells
• Changing medium for HEK293FT cells and HepG2 cells and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals

Aug 26:

• Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 60mm TC plates
• Changing medium of transfection of the previous day and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals

Aug 28:

• Changing medium of transfection of the previous day
• Staining and fixation of
• Drawing MTT standard curves

Aug 29:

• Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells
• Maintaining HEK293FT and HepG2 cell line

Aug 30:

• Staining and fixation for HEK cells transfected by pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Seeing the results for transfected HEK293FT cells: No obvious overlapping pattern of GFP signals and Mito-tracker
• Seeding HepG2 cells for Transfection of FABP construct and pEGFP-N1 in 96-well plate
• Seeding HEK 293FT cells for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Passaging HEK293FT cell line

Aug 31:

• Transfection with pEGFP-N1 and pCMV/PolyA/EGFP into HEK293FT cells

Sept 1:

• Checking results for transfection on Aug 31: pEGFP-N1 had strong signal while PolyA did not have signals

Sept 2:

• Transfection of FABP construct and pEGFP-N1 in HepG2 cells
• Check results for HEK293FT cells been transfected by pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP, by using confocal microscope

Sept 3:

• Adding FA into HepG2 cells been transfected by FABP construct and pEGFP-N1
• Maintaining HepG2 and HEK293FT cell line
• Drug selection Test on HEK293FT test by purmycin

Sept 4:

• Drug selection test on HEK293FT cells by neomycin and puromycin

Sept 5:

• Staining and fixation of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP and seeing he results by confocal microscope
• Making mew DMEM
• Drug selection test observation

Sept 6:

• Transfection of FAPB and pEGFP-N1 into HepG2 cells in 60 mm plates
• Maintaining cell line for HEK293FT cells

Sept 7:

• Checking results for transfection on Sept 6: pEGFP-N1 showed weak GFP signal, no signals for FABP
• Change medium for drug selection test

Sept 8:

• Maintaining cell line for HEK293FT cells and HepG2 cells

Sept 9:

• Checking transfection for FABP construct again: Weak signals were shown from the debris of the cells
• Transfection of AceA construct

Sept 11:

• Seeding HEK293FT cells into 35mm plates for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
• Cell splitting of ACE

Sept 12:

• Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells