2014/06/27

Transduction mouse cells (different incubation times)

2014/06/30

Transfection CHO cells with receptor

Transfection/ Virusproduction

Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21).

Viral Vectors - July

2014/07/03

Transfection CHO cells with receptor

CHO cells were transfected with the receptor (for later transduction). Medium was removed and filled with 2 ml new medium per well. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C. This time there were no results du to high density of cells during transduction.

Freezing (cryopreservation) of eukaryotic cells

Phoenix cells were frozen at -80°C.

• removal of medium and washing with cold PBS
• addition of 1 ml 0,05% Trypsin per plate, incubation for 1-2 min)
• stopping of reaction with 5 ml DMEM (with FCS)
• centrifugation (5 min, 900 rpm)
• removal of supernatant and resuspension in 2 ml FCS (+10% DMSO)
• quick transfer in steril cryotube (1ml per tube) and quick freezing in -80°C

2014/07/06

Transfection CHO cells with receptor

CHO cells were seeded on cover slips and transfected with the receptor (for later transduction). Due to the fact that cells must be in growth phase during transduction with virus the cell density was set to 40%. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

2014/07/08

Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21)

2014/07/10

Transfection of CHO cells with receptor

2014/07/10

Fixation of cells on cover slips

CHO cells (transfected with SLC7a1; 2014/07/04) were fixed on cover slips

• Medium was removed and cells were washed with PBS
• Appropriate amount of 4% PFA/PBS was added (200µl on 24W) and incubated for 10 min on ice
• PFA was removed and plate was washed with PBS
• Cover slips were fixed with Mowiol on slides

2014/07/11

Transfection of HEK cells with receptor

HEK 293 cells were transfected with the receptor (for later transduction). Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

2014/07/17

Transduction of mouse cells

green: anaylsis with flow cytometry

yellow: fixation with PFA on cover slips

• Removal of medium
• Washing with cold PBS
• Adding of 400 µl PFA and incubation for 10 min on ice, another 10 min at RT
• Incubation of cover slips for 10 sec in DAPI solution (1:5000 in water)
• Washing in water
• Mounting with Mowiol on slides

Cells detach from the cover slip, therefore a coating is necessary e.g. with Poly-L-Lysine  no results (better use poly-lysine for better grip of cells on cover slip)

blue: preparation for Western Blot via RIPA Lysis (as positive control for anti-CAT1 antibody)

• Removal of medium
• Washing with ice cold PBS
• Addition of 100 µl RIPA Buffer (completed with Phosphatase-Inhibitor-Mix)
• Incubation 10 min on ice
• Removal of cells with tip and transfer into Eppendorf tube
• Incubation for 10 min on ice
• Centrifugation for 5 min 10000 x g
• Transfer of 60 µl supernatant in new tube
• Addition of 15 µl 5 x SDS loading dye (with β-Mercaptoethanol)
• Cooking for 10 min at 95°C or for 15 min at 72°C
• Freezing at -24°C

Transfection of CHO cells with receptor

CHO cells growing in completed HTS medium (K1) were compared to CHO cells growing in completed DMEM medium. Cells were transfected with the receptor. Afterwards both kinds of CHO cells were infected with MuLV IRES EGFP and analyzyd using flow cytometry investigate which cells are better for transfection and transduction. Medium was changed after 5 h. Cells were incubated for 24 h at 37°C.

2014/07/21

Transfection/ Virus production

Phoenix cells were transfected with pMIG IRES EGFP and pMIG EGFP(protocol: 2014/05/21)

2014/07/31

Improvement of Transduction

Transduction of NIH3T3 cells with two different viral supernatants (MuLV IRES EGFP or MuLV EGFP) with either 1 or 2 µl Polybrene.

• 1. 1 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant
• 2. 2 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant
• 3. 2µl Polybrene was added to 1 ml viral supernatant; mixture was added to 1 ml DMEM

Centrifugation at 37°C for 45 min at 400 RPM. Results indicate that cells do not like to be centrifuged.

Testing difference in transfection efficiency of Phoenix and HEK cells/ negative control for MuLV

Viral Vectors - August

2014/08/01

Generation of a GFP mouse cell line

For testing whether MuLV can stable transfer genes into cells, a stable mouse cell line using this virus was generated.

Therefore two 100mm plates were transduced with 3 ml virus supernatant (MuLV IRES EGFP) and splitted as usual. Cells were sorted with a cell sorter. Analysis via FACS happened before and after sorting. The analysis was repeated after several rounds of splitting.

2014/08/03

Testing different transfection methods with different cells

For optimizing transfection in different cell lines transfection methods and different concentrations of the transfection mixtures were compared. The experiment was done with mouse cells (NIH3T3), hamster cells (CHO) and human cells (Phoenix). As transfection reagents lipofectamin and PEI were used in different concentrations.

Transfection with Lipofectamin (for 3 wells):

• (solutions A) 50 µl OptMEM was mixed with either:
• 1. 1 µl Lipofectamin + 1,5 µl PHB308
• 2. 2,5 µl Lipofectamin + 1,5 µl PHB308 or
• 3. 4 µl Lipofectamin + 1,5 µl PHB308
• incubation for 25 min at RT
• (solution B) 150 µl OptiMEM was mixed with 1,5 µl PHB308 (2,5 µg/µl) and 4 µl Plut Reagent
• incubation for 15 min
• solutions A (1-3) were then mixed with 50 µl of solution B and incubated for 5 min at RT
• 100 µl of transfection solution were added to each well
• no medium changing, incubation at 37°C for 24 h

Transfection with PEI (for 1 well):

• 0,2 µl PHB308 was mixed with 40 µl OptiMEM and DNA, for each well another concentration of DNA was added:
• 1. 1,5 µl PEI
• 2. 3 µl PEI
• 3. 5 µl PEI
• Incubation for (optimal) 10,8 min
• addition of 40 µL solution to each well
• medium changing after 5 h of incubation at 37°C, incubation for 24 h at 37°C

2014/08/06

Transfection of HEK cells with receptor

Transfection HEK and Pheonix cells with receptor (transfection while seeding)

2014/08/09

Virus dilution

Viral Vectors - September

Viral Vectors - October