1. Split the cells 1:5 into 10 cm plate (on 21.04.17)
2. Thaw PEI reagent at RT
3. Dilute plasmid in 1 ml serum free DMEM (2.5µg from each plasmid)
4. Add 15 µl PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
5. incubate 15 min at RT
6. Add the mix to the culture

1. Split cells 1:5 to a 6-well plate: 100 µl/well (on 12.05.17)
2. For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM
3. Establish Mastermix
4. Add 9 µg PEI to each mix while vortexing (PEI:DNA=3:1)
5. 15 min incubation
6. Add the Mix to the cells: 275.25 µl GFP-Mix or 215.5 µl to corresponding samples
7. After 48h: Fluorescence Microscopy

1. Split cells 1:5 to a 6-well plate: 330 µl (from 10 cm plate)/well (22.05.17)
2. For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM
3. Establish Mastermix
4. Add PEI to each mix while vortexing (PEI:DNA=3:1)
5. 15 min incubation
6. Add the Mix to the cells
7. After 48h: Fluorescence Microscopy & Flow Cytometry

1. pGFP: 2.5 µg/well
2. Mix I: 150 µl serum free DMEM and 6 µl LTX reagent
3. Mix II: 700 µl SERUM FREE DMEM + 2.5 µg pGFP + 14 µl PLUS reagent
4. Mix 150 µl Mix I with 150 µl Mix II
5. 5 min incubation at RT
6. Add 300 µl to cells

Cell lines: HPB-ALL, HUT78 and Jurkat Plasmid: pIG17_009 (GFP plasmid)

1. Count the cells
2. Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min
3. Resuspend the cell pellet with 100 µl P3 Buffer
4. Add 3 µg pIG17_009
5. Transfer the mixture into cuvette
6. BioRad setting: 250 V, 960 µF, 35 sec.
7. Add 500 µl RPMI medium to the cells immediately after electroporation
8. Plate the cells into 12-well plate with 2 ml Medium
9. Over night culturing
10. Wash the cuvettes for reusing

Plate 4 mio. Hek cells in 10 cm plate. (Will be around 7-8 mio cell the next day)
(optional: 5-6 mio. Hek cells in 15 cm plate)

Transfection of HEK cells for virus production.

• Packaging Mastermix

• PEI Mastermix

Cell lines: HUT78 and Jurkat Plasmid: pIG17_008 (mCherry plasmid)

1. Count the cells
2. Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min
3. Resuspend the cell pellet with 100 µl P3 Buffer
4. Add 3 µg pIG17_008
5. Transfer the mixture into cuvette
6. Amaxa Programm: X-005
7. Add 500 µl RPMI medium to the cells immediately after electroporation
8. Plate the cells into 12-well plate with 2 ml Medium
9. Over night culturing
10. Wash the cuvettes for reusing

• Collect the medium (containing virus supernatant) and add new medium after 24h

Cell lines: HUT78 and Jurkat Plasmid: pIG17_009 (GFP plasmid)

1. Count the cells
2. Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min
3. Resuspend the cell pellet with 100 µl P3 Buffer
4. Add 3 µg pIG17_009
5. Transfer the mixture into cuvette
6. BioRad setting: 250 V, 960 µF, 35 sec.
7. Add 500 µl RPMI medium to the cells immediately after electroporation
8. Plate the cells into 12-well plate with 2 ml Medium
9. Over night culturing
10. Wash the cuvettes for reusing

• Collect the medium (containing virus supernatant) and add new medium after 48h
• Mix with medium collected after 24h

• negative controls: Hut78 and Jurkat cells.
• positive control: GFP_Jurkat(p526)
• Amaxa Samples: Hut78 (mCherry and GFP) and Jurkat (mCherry)
• BioRad Samples: Hut78 (GFP) and Jurkat (GFP)

1. Count the cells
2. resuspend cell pellet (about 1 mio.) with FACS Buffer (PBS with 1%FCS)

1. Plate about 200000 cells for each wells.
2. Incubate the plates over weekend in incubator.

• Cell lines: Hut78
• Plasmid: pIG17_008 (mCherry plasmid)

1. After 72h → Change the medium of the plates (for suspension cells: first centrifuge and then resuspend cell pellet with new medium)
2. Incubation over night for FACS

no positive population

with ration of 1:1 (Medium vs. Virus medium) cells were most frequently infected and viable.

• Cell lines: Jurkat & Hut78
• Plasmid: pIG17_009
• Cell count: 1 mio. (Jurkat) 4 mio. (Hut78)
• Settings:

• Cell lines: Jurkat & Hut78
• Plasmid: pIG17_009
• Cell count: 1 mio.
• Program: X-005
• Flow cytometry after 24h.

No positive cell population.

no positive cell population.

• Jurkat(p526): mycoplasma positive
• Hut78(p526):mycoplasma positive
• HEK(p526): mycoplasma negative

No cell sorting, stock cultures should be sent for test.

• Cell lines: Jurkat
• Plasmid: pIG17_009
• Cell count: 2 mio./ approach
• 4 approaches:

4 approaches:

1. 1 transfection with pIG17_009
2. 2 transfections with pIG17_013
3. 1 untransfected as control

1. Split cells 1:5 to a 6-well plate: 100 µl/well (on 16.06.17)
2. For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM
3. Establish Mastermix
4. Add 9 µg PEI to each mix while vortexing (PEI:DNA=3:1)
5. 15 min incubation
6. Add the Mix to the cells
7. After 48h: Fluorescence Microscopy & Flow cytometry

• Cell: via PEI transfected HEK cells.
  
• CoCl2 stock: 1M
  
• End concentration of CoCl2: 100µM
  
• Cell observation under fluorescence microscopy after 2h:
  

-By adding CoCl2, the HRE-GFP transfected cells showed less fluorescence signal than without CoCl2…

• Flow cytometry: no expected signal

• Cell lines: Jurkat
• Plasmid: pIG17_009
• Cell count: 2 mio./ approach
• Setting: 96 well plates, well set 1(ABCD1)
• 4 approaches:

• Cell lines: Jurkat
• Plasmid: pIG17_009
• Cell count: 2 mio./ approach
• Setting: 96 well plates, well set 1(ABCD1), Expotential
• 4 approaches:

• HEK293T: mycoplasma negative
• Jurkat: mycoplasma negative
• Hut78: mycoplasma positive → abort culturing of Hut78!

• Not successful

• In total: 3*6 wells
• Transfection with pIG17_009 as positive control: 1 well/plate
• Transfection with pIG17_013: 4 well/plate

1. Split the cells 1:5 into 10 cm plate (on 23.06.17)
2. Thaw PEI reagent at RT
3. Dilute plasmid in 1 ml serum free DMEM (3µg from each plasmid)
4. Add 15 µl PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
5. incubate 15 min at RT
6. Add 200µl mix to wells

• Cell: 1 plate of via PEI transfected HEK cells. (from 24.06)
  
• CoCl2 stock: 1M
  
• End concentration of CoCl2: 100µM
  

* Cell observation under fluorescence microscopy every hour after CoCl2 treatment

• Flow cytometry after 8 hours

• Cell lines: Jurkat
• Plasmid: pIG17_009
• Cell count: 2 mio./ approach
• Setting: 12 well plates, well set 1
• 3 approaches:

• Flow cytometry after 24h.

1.Plate

2.Plate

No induction of GFP expression under hypoxia condition Problems:

• Cells stayed attached to the ground of plates even after treating with trypsin
• Cells clumps instead of single cell were detected

By increasing the amount of plasmid DNA, some cells were potentially transfected!!!

9µg for each approach

• Setting: 12 well plate, 1 well set, 1 pulse

Transfection of HEK cells for virus production.

• Packaging Mastermix

• PEI Mastermix

• Collect the medium (containing virus supernatant) and add new medium after 24h

• Collect the medium (containing virus supernatant) and add new medium after 24h
• Mix the virus medium (24h + 48h)

1. Plate about 200000 cells for each wells.
2. Incubate the plates over weekend in incubator.

• Prepare 2*6-well Plates of HEK cells
• Transfect cells via PEI:

9µg for each approach

• Setting: 12 well plate, 1 well set, 1 pulse

• Cells treated with CoCl2 built clumps, which depends on the incubation time and CoCl2 concentrations.

No induction of GFP expression under hypoxia condition

• Add different concentrations of CoCl2 (50µM, 100µM and 200 µM)
• Observe the cells under fluorescence microscope 3h, 6h, 8h, 16h, 18h and 24h after CoCl2 treatment
• No detectable induction of gene expression for every approach

• Both negative and positive controls showed abnormal populations → need to repeat the experiments.

No induction of GFP expression under hypoxia condition

• Add different concentrations of CoCl2 (50µM, 100µM and 200 µM)
• Observe the cells under fluorescence microscope 3h, 6h, 8h, 16h, 18h and 24h after CoCl2 treatment
• No detectable induction of gene expression for every approach

• Change the medium and add puromycin for selection

9µg for each approach

• Setting: 12 well plate, 1 well set, 1 pulse

• In total: 3*6 wells
• Transfection with pIG17_009 as positive control: 1 well
• Transfection with pIG17_022: 4 wellS
• Transfection with pIG17_023: 4 wellS
• Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI

1. Split the cells 1:5 into 10 cm plate (on 03.07.17)
2. Thaw PEI reagent at RT
3. Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)
4. Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
5. incubate 15 min at RT
6. Add 200µl mix to wells

• Cells transfected with pIG17_082 showed high transfection efficiency
• Cells transfected with our stock pIG17_009 showed low efficiency and low survival rate

• Plate about 4 mio. cells, make replicate

• No significant induction

Transfection of HEK cells for virus production.

• Packaging Mastermix

• PEI Mastermix

• Change the medium

4 Approaches (9µg/Approach) Setting: 12 well plate, 1 well set, 1 Pulse, ∞ Resistance

1. Electroporated without plasmid
2. CMV-GFP from Nicole (700 ng/µl)
3. CMV-GFP (Yael, 06.06.17, 140.4 ng/µl)
4. CMV-GFP (Dennis, 27.06.17, Dennis, 86.8 ng/µl)

• Collect virus medium
• Add new medium

• Collect virus medium
• Add new medium

• In total: 3*6 wells
• Transfection with pIG17_009 as positive control: 1 well (plasmid from Nicole); 1 well (Culture from Nicole, from us preped using promega miniprep)
• Transfection with pIG17_022: 5 wellS
• Transfection with pIG17_023: 5 wellS
• Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI

1. Split the cells 1:5 into 10 cm plate (on 03.07.17)
2. Thaw PEI reagent at RT
3. Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)
4. Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
5. incubate 15 min at RT
6. Add 200µl mix to wells

• Use 6-well plate
• plate 200000 cells/well
• Make Titer test with virus medium

The cells were not successfully infected.

Transfection of HEK cells for virus production.

• Packaging Mastermix

• PEI Mastermix

• In total: 3*6 wells
• One well with non-transfected cells
• Transfection with CMV-GFP as positive control: 1 well (JetStar); 1 well (Promega Mini)
• Transfection with pIG17_013: 4 wellS
• Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI

1. Split the cells 1:5 into 6-well plate (on 15.07.17)
2. Thaw PEI reagent at RT
3. Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)
4. Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
5. incubate 15 min at RT
6. Add 200µl mix to wells

• with 1%O2

• BioRad didn't work

• Fluorescence microscopy
• After 24h incubation: cells showed normal morphology and there was no autofluorescence.

• wash the virus-transducted cells (pIG17_119)

• In total: 2*12 wells
• One well with non-transfected cells
• One well: Transfection with CMV-GFP as positive control
• Transfection with pIG17_022: 5 wellS
• Transfection with pIG17_023: 5 wellS
• Transfection with pIG17_037: 5 wellS
• Transfection with pIG17_086: 5 wellS
• Per Approach: 1.5µg DNA, 100µl serum free DMEM and 4.5µg PEI

1. Split the cells 1:10 into 12-well plate (on 17.07.17)
2. Thaw PEI reagent at RT
3. Dilute plasmid in 100µl serum free DMEM (3µg from each plasmid)
4. Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
5. incubate 15 min at RT
6. Add 100 µl mix to wells

• In total: 4*6-well plates
• One well in each plate: with non-transfected cells
• One well in each plate: Transfection with CMV-GFP as positive control
• Transfection with pIG17_013: 8 wellS
• Transfection with pIG17_031: 8 wellS

1. Split the cells 1:10 into 6-well plate (on 17.07.17)
2. Thaw PEI reagent at RT
3. Dilute plasmid in 100µl serum free DMEM (3µg from each plasmid)
4. Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
5. incubate 15 min at RT
6. Add 200 µl mix to wells

• for 20 ml virus medium, use 5ml 20% sucrose
• carefully add virus medium to sucrose (very tricky)
• centrifuge at 4000 rpm over night
• After centrifugation: remove the medium, put the falcon tube upside down and air dry.
• slowly add 100 µl PBS (20-30x, also very tricky)
• Incubate the pellet for 30-60 min
• resuspend the cells 2x
• aliquot the virus
• store in -80°C freezer

• plate 200000 Jurkat cells/ well in one 6-well plate
• add the concentrated virus (pIG17_119 and pIG17_121) (30µl and 50µl)

• Treatment with 2*6 well plate: 1 with pIG17_013 and 1 with pIG17_031
• Make new 2M CoCl2 stock
• add CoCl2 to the cells, end-concentration: 0, 300µM, 500µM and 100µM
• Incubation for 24h and track the induction of cell expression

• Fluorescence microscopy
• After 48h incubation: cells were mostly dead showing autofluorescence.

• For mCherry excitation: FL3!!!

• Treatment with 2*6 well plate: 1 with pIG17_013 and 1 with pIG17_031
• Make new 2M CoCl2 stock
• add CoCl2 to the cells, end-concentration: 0, 300µM, 500µM and 100µM
• Incubation for 24h and track the induction of cell expression

• Fluorescence microscopy
• After 72h incubation: cells were mostly dead showing autofluorescence.

PEI Transfection of HEK cells for virus production.

• Packaging Mastermix

• PEI Mastermix

there was GFP positive signals during flow cytometry measurement but the cells were mycoplasma positive

• Collect virus

• Collect virus

• Both Cas9-GFP-gRNA5 and Cas9-GFP-scramble transfected cells were mycoplasma positive

1. Split the cells 1:2 in 12-well plate (on 24.07.17)
2. Per Approach: 1µg DNA + 8µg PEI and fill up to 100 µl with serum free DMEM
3. Thaw PEI reagent at RT
4. Add PEI(1µg/µl) to the mix while vortexing
5. incubate 15 min at RT
6. Add the mix to cells

• 3h after PEI transfection: remove media, add tetracycline to new media (1:1000 dilution)
• Tetracycline Stock: 2mg/ml (End: 1µg per well of 12-well plate)
• 3h after adding tetracycline: no induction
• incubation till the next day: microscopic observation
• Gene expression was induced by tetracycline compared to the tetracycline-untreated cells

1. Split the cells 1:5 in 12-well plate (on 24.07.17)
2. Per Approach: 1.5µg DNA + 4.5µg PEI and 100 µl serum free DMEM
3. Thaw PEI reagent at RT
4. Add PEI(1µg/µl) to the mix while vortexing
5. incubate 15 min at RT
6. Add the mix to cells

• Add corresponding concentration of CoCl2 to (hypoxic plasmid) transfected cells (CHO and HEK)
• CoCl2 stock: 2M

- gather 200 µl of cell supernatant
- incubate cell supernatant at 65°C for 30min
- centrifuge cell supernatant for 1 min at 1250g
- add 150 µl supernatant into a cuvette
- add 500 µl 2x SEAP buffer
- add 100 µl pNPP and remove bubbles carefully
- measure in a nano drop every 5min for 2h

• New HEK cells were mycoplasma negative

• Per Approach: 9µg DNA, 2 mio.JK cells

• Per Approach: 1.5µg DNA, 4.5µg PEI, 100µl DMEM

Due to the concentrations and volumes in the tubes for each approach two were used. Per construct 24 wells were used. Each well will have different CoCl2 concentrations. For each approach triplicates will be analysed.

For the knockout Kit we could see the most living GFP positive cells (26.2%) for the cotransfection of 203 and 104. The Cas9 GFP guide RNA5 had 5.21% GFP positive cells under the gated living cells.

1:10 dilution of a 2M CoCl2 stock with PBS. CoCl2 was added to HRE-SEAP approaches in order to induce the promoter, as well as to CMV-SEAP approaches

Approaches: For each approach triplicates were done.

Follow the protocol from Frederike (AG Schamel) PEI Transfection of HEK cells for virus production.

1. Prepare packaging and PEI mix separately and incubate for 10 min
2. Mix packaging mix with transferplasmid
3. Add PEI mix to DNA mix and incubate for 15 min
4. Add the PEI+DNA mix to cells

• Packaging Mastermix

Follow our protocol of PEI transfection

1. DNA:PEI ratio = 1:8
2. Mix DNA with 1 ml serum free DMEM
3. Add PEI while vortexing
4. 15 min incubation

- gather 200 µl of cell supernatant
- incubate cell supernatant at 65°C for 30min
- centrifuge cell supernatant for 1 min at 1250g
- add 80 µl supernatant into a 96 well plate
- add 100 µl 2x SEAP buffer
- add 20 µl pNPP and remove bubbles carefully
- measure in the plate reader every 30s for 2h

–> Experiment failed and will be repeated

• Plate 200000 Jurkat cells/well in 6-well plate
• Titer Test of virus
• Incubation during weekend

• testing of untransfected JK cells and the scramble JK cells that have been transfected with the KO Kit (for each 5 conditions will be tested)
• dilution of the Puro stock (10 mg/ml) 1:100 –> 100 µg/ml
• cells will be distributed in a 12 well plate with 1 ml of media

• cells will be distributed in a 12 well plate on the next day
• one master mix for PEI
• 1 ml DMEM, 30 µl PEI, 10 µg DNA per each approach
• cotransfection of CMV-SEAP (95%) and HRE-SEAP (95%) with CMV-GFP (5%)

• Per Approach: 9µg DNA, 2 mio JK cells

• GFP was observed under the microscope
• the cells were evenly distributed onto 12 well plates
• induction with CoCl2 after the cells recovered from the transfer
• 8 conditions per approach, everything done in triplicates

pei_lentivirus_production_08.08.17.xlsx

The SEAP assay was negative. No changes in the OD for either CMV-SEAP or HRE-SEAP could be observed. Due to these troubles the next test will be postponed till all constructs have undergone another test digestion and sequencing.

• Virus: pIG17_082, pIG17_119, pIG17_121 and pIG17_133
• Plate 200000 virus for infection
• As control: HEK with 1:1 infection

• Cas9-GFP-scramble

• Cas9-GFP-gRNA1

• Cas9-GFP-gRNA4

• Cas9-GFP-gRNA5

• 9µg DNA and 2 mio. cells/ approach

Day 6 after addition of puromycin: due to low cell numbers, the cells were spun down, resuspended in 100 µl and then counted.

Results for untransfected Jurkat cells and Jurkat cells transfected with the scramble:

Approaches:

• 1x scramble no treatment
• 1x scramble 0.2 µg/ml
• 2x KO Kit 203+101 0.2 µg
• 2x KO Kit 203+104 0.2 µg

Transfer to a 6 well plate

Due to low cell counts the duplicates were pooled together and the cells transferred to a 12 well plate.

in 10cm plates:

• 10 µg DNA
• 1ml DMEM
• 30 µl PEI

approaches:

• HRE-SEAP
• pWW56 as a positive SEAP control
• untransfected control

• the cells were evenly distributed onto 12 well plates
• induction with CoCl2 after the cells recovered from the transfer
• 8 conditions per approach, everything done in triplicates

Due to low cell counts the samples were transferred to a conical 96-well plate

• Approached by Frederike (AG Schamel)
• 2 Approaches (pIG17_119 & pIG17_121) with our HEK cells, envelope & packaging plasmid,PEI
• 1 Approach (pIG17_119) with material from AG Schamel: HEK, envelope & packaging plasmid, PEI

The SEAP assay was performed as mentioned before, however, there was a 1:5 dilution included of the HRE-SEAP samples.

For the analysis of the SEAP assay, the averages of the triplicates were calculated and the respective wild-type control subtracted from the HRE-SEAP samples.

For the undiluted HRE-SEAP construct the following graph was obtained:

For the 1:5 dilution the following graph was obtained:

For the 1:5 dilution absorbance values are missing at the start because they were negative and therefore regarded as 0.

The positive control did not work due to the promoter of this construct which was dependent on an input that wasn't given. Furthermore, the samples did not show what we expected, considering that the uninduced control showed a steeper slope than most of the samples. Due to this reason the experiment was repeated.

• Approaches for Titer test: both normal transduction and spin-infection.
• On 8/20: After washing the cells, there was no signal from reporter gene expression.

• Transferplasmid: pIG17_130 (duplicate)

transfection_pei_lentivirus_23.08.17.xlsx

• Add 8 ml 20% Sucrose (in PBS + 1Mm EDTA) in 50 ml Falcon Tube
• Add virus media slowly over sucrose (Use 25ml pippet)
• Centrifuge over night at 4000 rpm, 4°C

• Plate 200000 cells/well (HEK & Jurkat) into 6 well plate
• Titer of virus: 0, 15 µl, 30 µl

As shown in the picture the transduction of HEK cells showed high efficiency. The cells were also mycoplasma-negative, so that the cells can be sorted at Bioss.

The efficiency of transduction was quite low. The positive signal showed above may come from dead cells. New transduction was done on 8/30 with higher virus titer (45µl and 60µl).

Lentiviral transduction of Jurkat cells with higher virus titer showed higher efficiency than previous try. These cells are sent for microplasma-test and for cell sorting.

pei_transfection_of_hek_virus_30.08.17.xlsx

pei_transfection_of_hek_virus_2.09.17.xlsx

9µg for each approach

• Setting: 12 well plate, 1 well set, 1 pulse

Washing of the electroporated cells

Sorting of the electroporated cells Gating after:

• living cells
• single cells
• mCherry positive cells

Sorting strategy:
Control igem_gfp_mcherry_jurkat_elektro_tube_001_06092017102918.pdf
pIG17_031 igem_gfp_mcherry_jurkat_31_tube_001_06092017103019.pdf
pIG17_034 igem_gfp_mcherry_jurkat_34_tube_001_06092017110102.pdf
pIG17_037 igem_gfp_mcherry_jurkat_37_tube_001_06092017112901.pdf

Sorting reports:
130 Jurkat: sort_report_06092017122940.pdf
pIG17_034 sort_report_06092017112206.pdf
pIG17_037 sort_report_06092017120252.pdf

Induction of the promoters

- in a 96 well plate with 200 µl volume:

• used concentration of CoCl2: 20 mM
• approximately 15000 cells per approach

.

- in a 96 well plate with 200 µl volume:

• used concentration of NaOH: 1 M
• used concentration of lactate: 0.6 M
• used concentration of Forskolin: 10 mM
• approximately 15000 cells per approach

• 6 well plate with 3ml RPMI and the same concentration of lactate or NaOH as in the 96 well plate but without cells
• used in order to measure the pH
• amount of cells will be neglected as it is rather low

- in a 96 well plate with 200 µl volume:

• used concentration of VEGF: 1 µg/ml
• approximately 15000 cells per approach

transfection_pei_11.9.17.xlsx

Jurkat cells with CMV-GFP; transfection mix:

1. 12 well plate
2. 2 mio cells per well in 500 µl medium
3. 0.5 µg DNA per well
4. Viafect/DNA = 2:1 and 3:1
5. 50 µl total volume per approach (filled up with serum free DMEM)

• removal of the medium from pelleted cells
• resuspending of pelleted cells in the transfection mix
• addition of 500 µl RPMI 1649 with FCS after
  1. 0 seconds
  2. 4 seconds
  3. 8 seconds
  4. 16 seconds
  5. 32 seconds
• transfer of the cell suspension to a 24 well plate

Analysis in the fluorescence microscope showed that this method did not work again

Constructs used: pIG17_130, CRE 4x
transfection_pei.pdf

Constructs used: NFAT 4x, HRE 4x
transfection_pei_20.9..pdf The concentrated viruses were stored in -80°C freezer as backup.

The set up for the SEAP assay was the same as before, however there wild-type controls with the same amount of CoCl2 included.
All samples were done in triplicates

Induction with CoCl2, the used concentrations of CoCl2 were: 0, 20, 40, 80, 160, 320 and 640 µM.

The SEAP assay was performed as before, this time we included a cell count of each pooled triplicate.
For the analysis first the averages of the triplicates were calculated and then divided by the cell count. Afterwards, the corresponding wild-type control was subtracted from the samples and all negative values were set to 0.

We could see that only 20, 40 and 80 µM CoCl2 showed a steeper slope in our samples than the 0 µM control.

Constructs used: pIG17_134, CRE 4x
transfection_pei_lentivirus_24.9.17.xlsx

Constructs used: pIG17_133, Suiside plasmid transfection_pei_4.10.17.xlsx

Due to the problems with our GFP readout we performed a search in BLAST and found out that our eGFP was wrongly annotate and is a eCFP. We also could see that in the FACS. For this we tested our Jurkat knockdown 130 cells, that stably express CFP then. On the left these cells are measured in the CFP channel and on the right in the GFP channel. The depicted cells are gated for living and single cells:

There is a clear shift and our cells are really CFP positive and not GFP positive

With Forskolin and IBMX, the cAMP pathway in the cell is activated.

• Cell lines: WT Jurkat, stable CRE4X Jurkat
• Conditions: untreated, induction with 100 µM Forskolin, induction with 100 µM IBMX, induction with 100 µM Forskolin and 100 µM IBMX.

FACS:
Cells were gated for living, single, mCherry positive cells and then the amount of CFP positive cells was measured:

• On 11th of Oct.: PEI transfection with CMV-TDAG8 plasmid.

We obtained the following results after gating for living, single, mCherry positive cells: