labor:cell_culture - iGEM 2017

Lab Notebook Cell Culture

PEI transfection of HEK cells

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

05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

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

Plasmid number	component	concentration[ng/µL]	volume[µL] for 12 µg
pIG17_008	CMV_mCherry-delta-PstI_pA	471.5	25
pIG17_009	CMV_EGFP_pA	368.4	30

23.05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

Plasmid number	component
pIG17_008	CMV_mCherry-delta-PstI_pA
pIG17_009	CMV_EGFP_pA

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

Lipotransfection of Jurkat, HPB-ALL and HUT78 cells

Plasmid number	component
pIG17_009	CMV_EGFP_pA

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

30.05.17

BioRad electroporation

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

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

preparation for lentiviral transduction

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)

06.06.17

Production of Lentivirus

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_082	Transfer plasmid with GFP p526	253	25.4
pIG17_003	Envelope plasmid pMD2G	105	30.7
pIG17_004	Packaging Plasmid pCMV∆R8.74	150	21.5

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	30.63	45.03
pIG17_004	21.44	64.33
150mM NaCl	441.49	927.12
total	493.56	1036.476

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1 µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

07.06.17

Amaxa electroporation

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

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

Production of Lentivirus

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

BioRad electroporation

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

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

08.06.17

Production of Lentivirus

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

Flow Cytometry

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)

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

09.06.17

Lentiviral transduction

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

Jurkat sample	Medium [ml]	Virus [ml]
1	1	1
2	0	2

Hut78 sample	Medium [ml]	Virus [ml]
1	2	0
2	1.5	0.5
3	1	1
4	0	2

HEK sample	Medium [ml]	Virus [ml]
1	2	0
2	1.5	0.5
3	1	1
4	0	2

10.06.17

BioRad electroporation

Cell lines: Hut78
Plasmid: pIG17_008 (mCherry plasmid)

Component	Amount
Hut cells	3 mio. cells
pIG17_008	5 µg

Setting	Capacitance[µF]	Voltage	Cuvette size[cm]	Time Constance [msec]
Setting 1	250	150	0.2	4.0
Setting 2	500	120	0.2	10.1

12.06.17

Lentiviral transduction

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

Flow Cytometry for BioRad electroporation on June 10th

no positive population

13.06.17

Flow Cytometry for lentiviral infected cells

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

BioRad Electroporation

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

Cell type	Setting	Capacitance[µF]	Voltage	resistence	Time Constance [msec]
Jurkat	Setting 1	300	250	∞	35
Jurkat	Setting 2	350	200	1000Ω	35
Hut78	Setting 1	250	150	∞	50
Hut78	Setting 2	500	120	∞	50

14.06.17

Amaxa electroporation

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

Flow Cytometry for BioRad electroporated JK cells

No positive cell population.

15.06.17

Flow Cytometry for BioRad electroporated Hut78 cells and Amaxa electroporated T cells

no positive cell population.

16.06.17

Mycoplasma test(lentiviral transduced Jurkat and Hut78)

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

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

17.06.17

BioRad Electroporation with JK cells

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

Approach	Capacitance[µF]	Voltage	Cuvette size[cm]	Time Constance [msec]
1:negative control	960	250	0.2	35
2:condition 1	960	250	0.2	35
3:condition 2	960	250	0.2	35
4:condition 3	960	250	0.2	40

PEI transfection with HEK cells

4 approaches:

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

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

Plasmid number	component
pIG17_013	pHRE_Ptal_EGFP_pA

19.06.17

Hypoxia test (CoCl2)

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

BioRad Electroporation

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

Approach	Capacitance[µF]	Voltage	Cuvette size[cm]	Time Constance [msec]
1:negative control	960	250	0.2	35
2:condition1	960	250	0.2	35
3:condition2	960	250	0.2	30
4:condition3	960	250	0.2	40

20.06.17

BioRad electroporation

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

Approach	Capacitance[µF]	Voltage	Cuvette size[cm]	Resistance
1:negative control	950	250	0.2	1000Ω
2:condition1	350	250	0.2	∞
3:condition2	300	300	0.2	1000Ω
4:condition3	300	250	0.2	1000Ω

21.06.17

Mycoplasma-Test (Stock cultures)

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

Flow Cytometry (BioRad on Jun.19th)

Not successful

24.06.17

PEI transfection (pIG17_013) for hypoxia test

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

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

25.06.17

Hypoxia test (CoCl2)

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

Well	End concentration of CoCl2[µM]	Volume of Stock CoCl2[µl]
1: untransfected	100	200
2:pIG17_009 transfected	-	-
3:pIG17_013 transfected	-	-
4:pIG17_013 transfected	50	100
5:pIG17_013 transfected	100	200
6:pIG17_013 transfected	200	400

* Cell observation under fluorescence microscopy every hour after CoCl2 treatment

Flow cytometry after 8 hours

BioRad Electroporation

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

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1:3 µg plasmid	960	250	∞	20
2:6 µg plasmid	960	250	∞	20
3:9 µg plasmid	960	250	∞	20

Flow cytometry after 24h.

26.06.17

Hypoxia test (CoCl2)

1.Plate

Duration of Treatment	Well	End concentration of CoCl2[µM]	Volume of Stock CoCl2[µl]
6h	1: untransfected	100	200
6h	2:pIG17_009 transfected	-	-
6h	3:pIG17_013 transfected	-	-
6h	4:pIG17_013 transfected	50	100
6h	5:pIG17_013 transfected	100	200
6h	6:pIG17_013 transfected	200	400

2.Plate

Duration of Treatment	Well	End concentration of CoCl2[µM]	Volume of Stock CoCl2[µl]
24h	1: untransfected	100	200
24h	2:pIG17_009 transfected	-	-
24h	3:pIG17_013 transfected	-	-
24h	4:pIG17_013 transfected	50	100
24h	5:pIG17_013 transfected	100	200
24h	6:pIG17_013 transfected	200	400

Flow Cytometry (Hypoxia test & BioRad)

Hypoxia test

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

BioRad Electroporation

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

27.06.17

BioRad Electroporation

9µg for each approach

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

GFP Plasmid: pIG17_009

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	950	250	∞	20
2	950	250	1000Ω	20
3	950	250	1500Ω	20

Knock out

Approach	Plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	203 (Cas9) + 104 (gRNA)	950	250	∞	20
2	203 (Cas9)+ scramble	950	250	∞	20
3	sgRNA3 (gRNA_GFP)	950	250	∞	20
4	sgRNA3 (gRNA_GFP)	950	250	∞	20

28.06.17

Flow Cytometry: electroporation on June 27th

29.06.17

Production of Lentivirus

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_?	LentiCRISPR(AG Cathomen)	1100	5.85
pIG17_003	Envelope plasmid pMD2G	68	47.4
pIG17_004	Packaging Plasmid pCMV∆R8.74	209	15.4

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	47.4	99.54
pIG17_004	15.4	32.34
150mM NaCl	441.49	927.12
total	504.3	1059

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

30.06.17

Production of Lentivirus

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

01.07.17

Production of Lentivirus

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

Lentiviral transduction

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

Jurkat sample	Medium [ml]	Virus [ml]
1	2	0
2	1.5	0.5
3	1	1
4	0.5	1.5
5	0	2

PEI transfection with HEK for Hypoxia Test

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

well	plasmid
1	-
2	pIG17_009
3	pIG17_013
4	pIG17_013
5	pIG17_013
6	pIG17_013

BioRad Electroporation: Repeat pGFP transfection

9µg for each approach

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

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1: no plasmid	950	250	∞	20
2: Mock plasmid	950	250	∞	20
3: pIG17_009	950	250	∞	20
4: pIG17_009	950	250	1000Ω	20
5: pIG17_009	950	250	1500Ω	20

02.07.17

CoCl2 treatment with Jurkat cells

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

Repeat Hypoxia test (24h after PEI)

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

Flow Cytometry (BioRad on July 1st)

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

03.07.17

Repeat Hypoxia test (48h after PEI)

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

04.07.17

Lentiviral transduction (pLenti-CRISPR from AG Cathomen)

Change the medium and add puromycin for selection

BioRad Electroporation: Repeat pGFP transfection

9µg for each approach

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

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1: no plasmid	950	250	∞	20
2: Mock plasmid	950	250	∞	20
3: pIG17_009	950	250	∞	20
4: pIG17_009	950	250	500Ω	20
5: pIG17_082 (p526)	950	250	∞	20

05.07.17

PEI transfection (pIG17_022 and pIG17_023) for CTLA-4 promoter test

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

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

Flow Cytometry

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

06.07.17

Prepare cells for lentivirus-production

Plate about 4 mio. cells, make replicate

VEGF induction with Modeling group

No significant induction

07.07.17

Lentivirus(pIG17_119) production in HEK cells

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_119	pCRE_GFP_CMV_mCherry	167	38.5
pIG17_003	Envelope plasmid pMD2G	243.4	13.3
pIG17_004	Packaging Plasmid pCMV∆R8.74	819	4

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	13.3	28
pIG17_004	4	8.4
150mM NaCl	441.49	927.12
total	458.8	963.52

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

08.07.17

Lentivirus(pIG17_119) production in HEK cells

Change the medium

BioRad electroporation

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

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

09.07.17

Lentivirus(pIG17_119) production in HEK cells

Collect virus medium
Add new medium

10.07.17

Lentivirus(pIG17_119) production in HEK cells

Collect virus medium
Add new medium

11.07.17

PEI transfection (CTLA-4 Test)

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

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

Lentiviral transduction (pIG17_119) of Jurkat cells

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

The cells were not successfully infected.

12.07.17

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no plasmid	950	250	∞	20
2	CMV-GFP (Nicole)	950	250	∞	20
3	CMV-GFP (Culture from Nicole, promega mini)	950	250	∞	20
4	pIG17_104: Cas9-GFP-gRNA3	950	250	∞	20

14.07.17

Lentivirus(pIG17_121) production in HEK cells

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_121	pCTLA4(380)_GFP_CMV_mCherry	1682	3.82
pIG17_003	Envelope plasmid pMD2G	321.9	10
pIG17_004	Packaging Plasmid pCMV∆R8.74	819	4

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	10	21
pIG17_004	4	8.4
150mM NaCl	441.49	927.12
pIG17_121	3.82	8
total	504.95	1060

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no plasmid	950	250	∞	20
2	6µg Cas9-GFP-gRNA3	950	250	∞	20
3	9µg Cas9-GFP-gRNA3	950	250	∞	20
4	12µg Cas9-GFP-gRNA3	950	250	∞	20

15.07.17

collect virus (pIG17_121)

flow cytometry (BioRad 7/14)

There is no GFP positive cell population

16.07.17

PEI transfection

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

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

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no plasmid	950	250	∞	20
2	CMV-GFP JetStar	950	250	∞	20
3	CMV-GFP promega mini	950	250	∞	20
4	CMV-mCherry (Nicole)	950	250	∞	20
5	pIG17_008 (Jana)	950	250	∞	20

17.07.17

Hypoxia Test in hypoxia incubator (AG Cathomen)

with 1%O2

Flow Cytometry

BioRad didn't work

18.07.17

Hypoxia Test in hypoxia incubator (AG Cathomen)

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

Lentiviral transduction

wash the virus-transducted cells (pIG17_119)

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	pIG17_119	950	250	∞	20
2	pIG17_121	950	250	∞	20
3	pIG17_031	950	250	∞	20
4	pIG17_034	950	250	∞	20
5	pIG17_037	950	250	∞	20
5	pIG17_086	950	250	∞	20

PEI transfection (CTLA-4 Test)

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

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

PEI transfection (hypoxia test with CoCl2)

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

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

19.07.17

Concentrate the viruses (pIG17_119 and pIG17_121)

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

Lentiviral transduction

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

CoCl2 treatment (24h after PEI)

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

Hypoxia Test in hypoxia incubator (AG Cathomen)

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

Flow Cytometry (BioRad on July 20th)

For mCherry excitation: FL3!!!

20.07.17

CoCl2 treatment (48h after PEI)

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

Hypoxia Test in hypoxia incubator (AG Cathomen)

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

21.07.17

BioRad Electroporation (Knock-Out plasmids)

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	-	950	250	∞	20
2	CMV-GFP (#503 from Nicole)	950	250	∞	20
3	pIG17_082 (p526)	950	250	∞	20
4	Cas9-GFP-gRNA5	950	250	∞	20
5	Cas9-GFP-scramble	950	250	∞	20

22.07.17

Lentivirus(pIG17_119 and pIG17_121) production in HEK cells

PEI Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_119	pCRE_GFP_CMV_mCherry	167	38
pIG17_121	pCTLA4(380)_GFP_CMV_mCherry	1682	3.82
pIG17_003	Envelope plasmid pMD2G	105	30.7
pIG17_004	Packaging Plasmid pCMV∆R8.74	208	15.5

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	30.7	64.5
pIG17_004	15.5	32.6
150mM NaCl	441.49	927.12

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

22.07.17

Flow Cytometry: BioRad Electroporation 7/21

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

Lentivirus(pIG17_119 and pIG17_121) production in HEK cells

Collect virus

23.07.17

Lentivirus(pIG17_119 and pIG17_121) production in HEK cells

Collect virus

24.07.17

Mycoplamsa Test

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

25.07.17

PEI transfection of CHO cells: Hypoxia & Tet system

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

Plasmid	Approach	DNA Concentration[ng/µl]	DNA volume	Volume of DMEM[µl]
CMV-GFP (#503 from Nicole)	1	25.5	40	52
CMV-mCherry (AG Hiltbrunner)	1	1200	1	91
pIG17_012	6	253	24	528
pIG17_013	6	583.3	10.3	541.7
pIG17_031	6	746.9	8	544
Tet-GFP (Shima)	2 (protocol from Shima)	520	3.8	180.2
Tet-GFP (Shima)	2 (our protocol)	520	5.8	200

Tetracycline induction of CHO

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

PEI transfection of HEK cells: Hypoxia

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

Plasmid	Approach	DNA Concentration[ng/µl]	DNA volume
CMV-GFP (#503 from Nicole)	1	25.5	59
CMV-mCherry (AG Hiltbrunner)	1	1200	1.25
pIG17_012	6	131.4	12
pIG17_013	6	583.3	15.5
pIG17_031	6	746.9	12

26.07.17

CoCl2 treatment

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

Endconcentration of CoCl2[µM]	Volume from Stock[µl]
0	0
100	0.1
200	0.2
300	0.3
500	0.5
1000	1

27.07.17

SEAP assay with HEK cells

- 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

Replace Stock cultures with new culture from Toolbox

31.07.17

Mycoplasma Test: New HEK stock

New HEK cells were mycoplasma negative

01.08.17

BioRad Electroporation

Per Approach: 9µg DNA, 2 mio.JK cells

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	pIG17_009 (06.06 Yael)	950	250	∞	20
2	Cas9-GFP-gRNA5	950	250	∞	20
3	Cas9-GFP-scramble	950	250	∞	20
4	KO-Kit: 203+101	950	250	∞	20
5	KO-Kit: 203+104	950	250	∞	20
6	KO-Kit scramble	950	250	∞	20

PEI transfection: Hypoxia - SEAP assay in HEK cells

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

Plasmid number	component	concentrations of the used tubes[ng/µL]	volumes from the used tubes[µL] for 36µg
pIG17_013	HRE-SEAP	583.3 and 88.1	55.7 and 40
pIG17_002	CMV-SEAP	92.0 and 91.4	350 and 41.6

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.

02.08.17

Flow Cytometry of BioRad Electroporation (8/1): KO plasmids

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.

Addition of CoCl2: Hypoxia - SEAP assay in HEK 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.

CoCL2 concentration[µM]	volume[µL] of 200mM CoCl2
0	0
100	0.55
150	0.825
200	1.1
250	1.375
300	1.65
500	2.75
1000	5.5

Lentivirus production in HEK cells: Mock, Knock-down plasmid (114,115,116), pIG17_119, pIG17_121

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

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

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	No. of Approaches	concentration[ng/µL]	volume [µL]
pIG17_082	Mock(p526):EF1-GFP	2	68	189.1
pIG17_114	Knock-down plasmid	2	350; 432	18.4; 15
pIG17_115	Knock-down plasmid	1	-	all two tubes
pIG17_116	Knock-down plasmid	2	492; 356	20; 8.5
pIG17_003	Envelope plasmid pMD2G	7	150;68.7;63.7	30;180;80
pIG17_004	Packaging Plasmid pCMV∆R8.74	7	926.6	24.3

Packaging Mastermix

Component	7x [µL]
pIG17_003	290
pIG17_004	24.3
150mM NaCl	3090.43

Follow our protocol of PEI transfection

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

Component	Mass/approach[µg]
pIG17_003	3.2
pIG17_004	3.2
pIG17_119	6.4
pIG17_121	6.4

03.08.17

Hypoxia - SEAP assay in HEK cells

- 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

05.08.17

Lentiviral Transduction

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

Puromycin killing curve

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

concentration of Puro	volume from 100 µg/ml [µl]
0	0
0.125	1.25
0.2	2
0.25	2.5
0.5	5

06.08.17

PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay

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%)

construct	concentration [ng/µl]	volume added for 9.5 µg [µl]
HRE-SEAP	248.2	38.3
CMV-SEAP	415.2	22.9

construct	concentration [ng/µl]	volume added for 0.5 µg [µl]
CMV-GFP	303.1	1.6

Per Approach: 9µg DNA, 2 mio JK cells

BioRad Electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no Plasmid	950	250	∞	20
2	CMV-GFP	950	250	∞	20
3	CMV-GFP	950	250	∞	20
4	Ca construct	950	250	∞	20
5	Ca construct	950	250	∞	20
6	Ca construct	950	250	∞	20
7	Cas9-GFP-gRNA1	950	250	∞	20
8	Cas9-GFP-gRNA1	950	250	∞	20
9	Cas9-GFP-gRNA4	950	250	∞	20
10	Cas9-GFP-gRNA4	950	250	∞	20
11	Cas9-GFP-gRNA5	950	250	∞	20
12	Cas9-GFP-gRNA5	950	250	∞	20
13	Cas9-GFP-scramble	950	250	∞	20
14	Cas9-GFP-scramble	950	250	∞	20

07.08.17

PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay

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

concentration of CoCl2 [µM]	volume added [µl]
0	0
100	0,5
150	0,75
200	1
250	1,25
300	1,5
500	2,5
1000	5

08.08.17

Lentivirus production in HEK cells: Mock, pIG17_133, pIG17_119, pIG17_121

PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay

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.

10.08.17

Lentiviral transduction

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

Cell Sorting: KO-plasmid transfected cells

Cas9-GFP-scramble

Cas9-GFP-gRNA1

Cas9-GFP-gRNA4

Cas9-GFP-gRNA5

11.08.17

BioRad Electroporation

9µg DNA and 2 mio. cells/ approach

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no Plasmid	950	250	∞	20
2	KO Kit 203+101	950	250	∞	20
3	KO Kit 203+104	950	250	∞	20
4	Lenti-Cas9-Puro	950	250	∞	20

Puromycin killing curve

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:

Puromycin addition to the electroplated KO Kit cells

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

12.08.17

Puromycin: electroplated KO Kit cells

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

14.08.17

PEI: HRE-SEAP, pWW56

in 10cm plates:

10 µg DNA
1ml DMEM
30 µl PEI

approaches:

HRE-SEAP
pWW56 as a positive SEAP control
untransfected control

construct	concentration [ng/µl]	volume used for 10 µg DNA
HRE-SEAP	420,5	23,78
pWW56	555,5	18,00

15.08.17

SEAP

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

concentration of CoCl2 [µM]	volume added [µl]
0	0
100	0,5
150	0,75
200	1
250	1,25
300	1,5
500	2,5
1000	5

Electroporated KO Kit cells

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

BioRad Electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no Plasmid	950	250	∞	20
2	CMV-GFP + CMV-mCherry	950	250	∞	20
3	pIG17_086	950	250	∞	20
4	pIG17_086	950	250	∞	20

Lentivirus Production in HEK cells

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

16.08.17

SEAP

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.

17.08.17

Lentiviral Transduction with virus-medium

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.

23.08.17

PEI transfection of HEK cells for lentivirus production

Transferplasmid: pIG17_130 (duplicate)

25.08.17

Concentrate virus (pIG17_130)

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

26.08.17

Lentiviral Transduction (pIG17_130)

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.

30.08.17

PEI transfection of HEK cells for lentivirus production (133&134)

02.09.17

PEI transfection of HEK cells for lentivirus production (132)

04.09.17

BioRad Electroporation

9µg for each approach

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

Number of approaches	Plasmid	Concentration [ng/µl]	Volume for 9ng [µl]	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no DNA	0	0	950	250	∞	20
1	CMV GFP, CMV mCherry	327.1, 1200	1376, 3.75	950	250	∞	20
2	pIG17_031	746.9	12.05	950	250	∞	20
2	pIG17_034	1210.9	7.43	950	250	∞	20
2	pIG17_037	851.9	10.56	950	250	∞	20

05.09.17

Washing of the electroporated cells

06.09.17

Sorting of the electroporated cells Gating after:

living cells
single cells
mCherry positive cells

Sorting strategy:
Control
pIG17_031
pIG17_034
pIG17_037

Sorting reports:
130 Jurkat:
pIG17_034
pIG17_037

07.09.17

Induction of the promoters

Set-up for pIG17_031 - hypoxia

- in a 96 well plate with 200 µl volume:

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

CoCl2 concentration [µM]	Number of approaches	Volume added [µl]
0	1	0
50	2	0.5
100	2	1
200	2	2
400	2	4
800	2	8

.

Set-up for pIG17_034 - pH

- 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

Lactate (L), NaOH (N) or Forskolin (F) concentration or pH	Number of approaches	Volume added [µl]
100 µM (F)	1	2
8.17 pH (N)	2	0.4
7.85 pH	2	0
6.54 pH (L)	2	8
6.19 pH (L)	2	10.67

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

Set-up for pIG17_037 - VEGF

- in a 96 well plate with 200 µl volume:

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

VEGF concentration [ng/ml]	Number of approaches	Volume added [µl]
0	1	0
2.5	2	0.5
5	2	1
10	2	2
20	2	4
40	2	8

Set-up for pIG17_031 - hypoxia

07.09.17 Cell sorting

Lentiviral transducted HEK and Jurkat cells with hIF1a shRNA1 (pIG17_133) and hIF1a shRNA2 (pIG17_134)were sorted for GFP positive cells (excitation with laser channel FL1)

HEK hif1a shRNA1

HEK hif1a shRNA2

Jurkat Hif1a shRNA1

Jurkat Hif1a shRNA2

27.08.17 - 07.09.17

Neomycin killing curve of Jurkat cells

11.09.17

PEI transfection of HEK cells for lenvirus production

Viafect - new testing

Jurkat cells with CMV-GFP; transfection mix:

12 well plate
2 mio cells per well in 500 µl medium
0.5 µg DNA per well
Viafect/DNA = 2:1 and 3:1
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

15.09.17

PEI transfection of HEK cells for lenvirus production

Constructs used: pIG17_130, CRE 4x

20.09.17

PEI transfection of HEK cells for lenvirus production

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

21.09.17

PEI transfection of HEK cells for SEAP assay

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

21.09.17 Cell sorting

Lentiviral transducted HEK and Jurkat cells with HRE4x-GFP-CMV-mCherry and NFAT4x-GFP-CMV-mCherry were sorted for mCherry positive cells (excitation with laser channel FL3)

HEK HRE4x-GFP-CMV-mCherry

HEK NFAT4x-GFP-CMV-mCherry

Jurkat HRE4x-GFP-CMV-mCherry

Jurkat NFAT4x-GFP-CMV-mCherry

22.09.17

SEAP assay

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

22.09.17 Cell sorting

Lentiviral transducted HEK and Jurkat cells with CRE-GFP-CMV-mCherry were sorted for mCherry positive cells (excitation with laser channel FL3) and those were transducer with hif1a shRNA3 (pIG17_130) were sorted for GFP positive cells (excitation with laser channel FL1).

HEK CRE4x-GFP-CMV-mCherry

HEK hif1a shRNA3

Jurkat CRE4x-GFP-CMV-mCherry

Jurkat hif1a shRNA3

23.09.17

SEAP assay

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.

24.09.17

PEI transfection of HEK cells for lenvirus production

Constructs used: pIG17_134, CRE 4x

24.09.17

HRE testing with stable HEK cell lines

this experiment didn't work.

04.10.17

PEI transfection of HEK cells for lenvirus production

Constructs used: pIG17_133, Suicide plasmid

04.10.17 Cell sorting

Due to low viability of previous sorted wit hif1a shRNA3 transducer Jurkat cells, the lentiviral transduction was repeated.

Lentiviral transducted Jurkat cells with hif1a shRNA3 (pIG17_134) were sorted for GFP positive cells (excitation with laser channel FL1).

Jurkat hif1a shRNA2

07.10.17

CRE test in Jurkat

JK were tested in the pH 7.7, 7.1, 6.5 and 7.7 adjusted. Some samples were treated with forskolin as a positive control. Analysis was done in a FACS and gating was done for GFP positive cells that are alive and mCherry positive. All samples were performed in triplicates. Shown are GFP positive cells. JK were tested in the pH 7.7, 7.1, 6.5 and 7.7 adjusted. Some samples were treated with forskolin as a positive control. Analysis was done in a FACS and gating was done for GFP positive cells that are alive and mCherry positive. All samples were performed in triplicates. Shown are GFP positive cells.

09.10.17

CFP test

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

CRE Test in Jurkat cells

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

10.10.17

HRE Test in Jurkat cells

Analysis was done with a FACS. The usual gating strategy was applied.

HRE Test in HEK cells

Analysis was done with a FACS. The usual gating strategy was applied.

12.10.17

CRE Test in HEK cells

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

CRE Test in Jurkat cells

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

VEGF Test in Jurkat cells

13.10.17 Cell sorting

Lentiviral transducted HEK and Jurkat cells with kill-switch construct were sorted for GFP positive cells (excitation with laser channel FL1).

HEK kill switch

Jurkat kill switch

18.10.17

HRE Test in CHO cells

PEI transfection of HEK cells with plasmid HRE-CFP and HRE4x-CFP

Analysis was done with a FACS. The usual gating strategy was applied. Depicted are CFP positive cells (that are alive, single cells and mCherry positive)

21.10.17

CRE Test in HEK cells

PEI transfection of HEK cells with plasmid SV40-TDAG8

Team:Freiburg/Notebook Cell culture

Lab Notebook Cell Culture

PEI transfection of HEK cells

05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

23.05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

Lipotransfection of Jurkat, HPB-ALL and HUT78 cells

30.05.17

BioRad electroporation

preparation for lentiviral transduction

06.06.17

Production of Lentivirus

07.06.17

Amaxa electroporation

Production of Lentivirus

BioRad electroporation

08.06.17

Production of Lentivirus

Flow Cytometry

09.06.17

Lentiviral transduction

10.06.17

BioRad electroporation

12.06.17

Lentiviral transduction

Flow Cytometry for BioRad electroporation on June 10th

13.06.17

Flow Cytometry for lentiviral infected cells

BioRad Electroporation

14.06.17

Amaxa electroporation

Flow Cytometry for BioRad electroporated JK cells

15.06.17

Flow Cytometry for BioRad electroporated Hut78 cells and Amaxa electroporated T cells

16.06.17

Mycoplasma test(lentiviral transduced Jurkat and Hut78)

17.06.17

BioRad Electroporation with JK cells

PEI transfection with HEK cells

19.06.17

Hypoxia test (CoCl2)

BioRad Electroporation

20.06.17

BioRad electroporation

21.06.17

Mycoplasma-Test (Stock cultures)

Flow Cytometry (BioRad on Jun.19th)

24.06.17

PEI transfection (pIG17_013) for hypoxia test

25.06.17

Hypoxia test (CoCl2)

BioRad Electroporation

26.06.17

Hypoxia test (CoCl2)

Flow Cytometry (Hypoxia test & BioRad)

Hypoxia test

BioRad Electroporation

27.06.17

BioRad Electroporation

GFP Plasmid: pIG17_009

Knock out

28.06.17

Flow Cytometry: electroporation on June 27th

29.06.17

Production of Lentivirus

30.06.17

Production of Lentivirus

01.07.17

Production of Lentivirus

Lentiviral transduction

PEI transfection with HEK for Hypoxia Test

BioRad Electroporation: Repeat pGFP transfection

02.07.17

CoCl2 treatment with Jurkat cells

Repeat Hypoxia test (24h after PEI)

Flow Cytometry (BioRad on July 1st)

03.07.17

Repeat Hypoxia test (48h after PEI)

04.07.17