Team:Freiburg/Notebook Modeling

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Lab Notebook Modeling

June

06.06.17

Titration RPMI 1640 vs. lactic acid

Aim: setup of pH in the Medium for pCRE-tests

stock-solutions:
c(lactic acid, conc.) = 12.09 mol/l
c(lactic acid, 1:20) = 0.6 mol/l (diluted with RPMI 1640)

Execution:

1. add lactic acid (0.6 M) to medium (Table)
2. measure pH with pH-Meter
3. repeat each measurement 3 times

V(RPMI 1640) [µl]	V(lactic acid, 0.6 M) [µl]	c(lactic acid) [µmol/mL]	s(c ) [µmol/ml]	pH 1	pH 2	pH 3	pH	s(pH)
2000	20	5.99	0.2	7.14	7.18	7.16	7.16	0.01
2000	40	11.9	0.3	6.90	6.91	6.88	6.90	0.01
2000	60	17.6	0.5	6.57	6.61	6.66	6.61	0.03
2000	80	23.3	0.6	6.35	6.31	6.27	6.31	0.02
2000	100	28.8	0.8	5.91	5.65	5.75	5.77	0.08
2000	120	34.2	0.9	4.99	4.96	4.93	4.96	0.02
2000	140	40	1	4.41	4.37	4.36	4.38	0.02
2000	160	45	1	4.31	4.12	4.10	4.18	0.07
2000	180	50	1	3.98	3.95	3.98	3.97	0.01

Resulting Titration curve for lactic acid in RPMI 1640:

Titration curve with pH vs. c(lactic acid)	Titration curve with pH vs. V(lactic acid 0.6 M) per ml

13.06.17

Cell survival and GFP-stability at different pH-values

used cells: Jurkat with CMV_GFP
cell density: 1 mio cells/ml
incubation times: 3 h, 6 h, 12 h

Execution:

1. count cells
2. centrifuge cells
3. resuspend cell pellet in RPMI 1640 (target-concentration: 2 mio cells/ml)
4. mix 1.5 ml RPMI 1640 + calculated volume of lactic acid (0.6 M) + 500 µl cell suspension on 12-well-plate (Table 1)
5. incubate for defined time in the CO₂-incubator
6. centrifuge samples
7. resuspend cell pellets in PBS FACS buffer (500 µl)
8. FACS analysis
9. used medium was stored at 4°C for pH-test next day

pH	V(lactic acid, 0.6 M) [µl]	RPMI 1640 [µl]
5.0	120	2000
5.5	107	2000
6.0	92	2000
6.5	68	2000
7.0	0	2000

Results:

→ Jurkat cells can survive in medium with pH ≥ 6.0

cell survival

after 3h	after 6h	after 12h

GFP fluorescence

after 3h	after 6h	after 12h

14.06.17

pH-measurement of used medium (13.06.17)

set pH	6 h	12 h
pH = 5.0	4.67	4.78
pH = 5.5	5.19	5.43
pH = 6.0	6.26	6.33
pH = 6.5	6.95	6.76
untreated (pH = 7.25)	7.73	7.47

Result: pH in incubation medium is rising over the time! Expected was a lowering, as the cells secrete lactic acid.

17.06.17

Investigation of the pH-changes

test of pH-change of RPMI 1640 at different conditions

tested conditions:

• incubation in stock RPMI 1640 or RPMI 1640 with lactic acid (pH = 6.0)
• medium with/without Jurkat cells (500k/ml)
• incubation at 37°C in incubator with CO₂ (5%) atmosphere or incubated with cap
• incubation at atmospheric conditions
• incubation time: 6 h

1. make aliquots of RPMI 1640 (15 ml, pH = 6.0) and untreated RPMI 1640 (15 ml)
2. aliquot 1: measure pH before incubation
3. other aliquots: incubate at conditions described above
   

Results

6 h, 37 °C, 5% CO2	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.46	7.68

6 h, 37 °C, 5% CO2, Jurkat cells	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.48	7.70

6 h, 37 °C, with cap	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.63	8.46

6 h, 25 °C, atmospheric conditions	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.24	7.79

19.06.17

pH change vs. incubation time

1. set pH to 6.3
2. prepare 2 ml aliquots of RPMI 1640 in falcon tubes
3. incubate in CO₂ incubator for t (min) = 0, 10, 20, 30, 50
4. measure pH directly after taking sample out of incubator
5. measure pH one minute later

time /min	pH direct	pH after 1 min
0	6.32	6.32
10	6.03	6.25
20	6.06	6.28
30	5.88	6.36
50	6.36	6.65

pH is strongly rising after taking the sample out of the incubator. This may be caused by the solved CO₂, that lowers the pH in the incubator. After taking the sample out of the incubator, the CO₂ goes to the gas phase which causes a rising of the pH. Additionally, the bicarbonate buffer is destabilized as the addition of lactic acid moves the equilibrium of the buffer to CO₂ + H₂O. That leads to a higher pH than before the incubation.

July

04.07.17

VEGF dilution

m₀ = 10 µg start concentration: solid state target concentration: 1 µg/ml

1. resuspend pellet in 100 µl ddH₂O (stock solution, 100 µg/ml)
2. dilute 1 µl of stock solution with 99 µl ddH₂O (aliquots, 1 µg/ml)
3. store aliquots at -20 °C

06.07.17

pCTLA4 vs. [VEGF]

Aim: determination of suitable [VEGF] and incubation time

tested cells: HEK with transient pCTLA4-GFP (pIG17_022 (330 bp); pIG17_023 (380 bp)),PEI transfected
[VEGF](ng/ml) = 0.0, 0.5, 5.0, 10.0
t (h) = 3, 8, 24
controls: CMV_GFP (pIG_009), untransfected cells

Execution:

1. cells were PEI transfected by cell culture on 05.07.17
2. 24h after PEI transfection: replace old medium by RPMI 1640 (2 ml / well), add VEGF (1 µg/ml)
3. after incubation time: analysis via fluorescence microscope and FACS

Results:

18.07.17

PEI transfection of plasmids pIG17_022/23/37/86

used cells: HEK cells

Plasmid number	component
pIG17_022	Ctla4(380bp)-GFP
pIG17_023	Ctla4(330bp)-GFP
pIG17_037	Ctla4(330bp)-GFP-CMV-mCherry
pIG17_086	Ctla4(380bp)-GFP-CMV-mCherry

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 (2.5 µg from each plasmid)
   
4. Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
   
5. incubate 15 min at RT
   
6. Add the mix to the culture
   
7. incubate for 24 h
   

19.07.17

VEGF test

used cells: PEI transfected cells from 19.07.17 with pIG17_022/023/037/086

1. add following VEGF concentrations to the cells: 0 ng/ml, 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml
   
2. incubate for 24 h
   

fluorescence microscope:

construct	bright field	GFP
positive control CMV-CFP
negative control

August

05.08.17

Interlab Study transformation

- heat shock transformation of DH5-α with plasmids from Kit plate 7

well	approach
21B	positive control
21D	negative control
21F	Device 1
21H	Device 2
21J	Device 3
21L	Device 4
21N	Device 5
21P	Device 6

Interlab Study OD600 calibration measurements

- measure absorbance at 600 nm of H20 (100 µl) and LUDOX-S40 (100 µl) - each four replicates

replicate	LUDOX-S40	H2O
1	0,043	0,035
2	0,049	0,036
3	0,047	0,035
4	0,052	0,035

Interlab Study fluorescein standard curve measurements

- centrifuge fluorescein
- dilute fluorescein in PBS to a final concentration of 50 µM
- add PBS (100 µl) into well A2-A12, B2-B12, C2-C12, D2-D12 of a 96-well-plate
- add fluorescein stock solution (200 µl, 50µM) into well A1, B1, C1, D1
- transfer 100 µl from well A1 into well A2, pipett up and down
- transfer 100 µl from well A2 into well A3, pipett up and down
- do the same till the end of the row
- transfer 100 µl from every last well into the liquid waste
- repeat these steps for rpw B, C and D
- measure the fluorescence intensity in the plate reader

uM Fluorescein	50,00	25	12,5	6,25	3,125	1,5625	0,78125	0,390625	0,1953125	0,09765625	0,048828125	0
Replicate 1	41550	28587	15976	9537	5674	2712	1357	639	348	160	91	0
Replicate 2	42434	29060	17592	10169	5250	2705	1155	581	302	160	67	11
Replicate 3	42066	29363	17743	9882	5015	2671	1303	665	314	187	87	0
Replicate 4	40538	28766	17808	9934	5127	2818	1344	662	366	176	113	13

06.08.17

pick colonies from Interlab Study

- pick 2 colonies from each plate
- incubation overnight at 37°C

07.08.17

Interlab Study cell measurements

- measure OD600 of the overnight cultures
- dilute the bacteria in medium with Chloramphenicol to a final OD = 0.02
- incubation at 37°C and 220 rpm
- take 500 µl of the cultures after 0h, 2h, 4h and 6h and place samples on ice
- transfer four replicates of 100 µl of each sample into a well of a 96-well-plate
- measure OD600 and fluorescence intensity in the plate reader (settings: excitation: 485 nm; emission: 530/30)

Abs600 after 0h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,044	0,042	0,045	0,045	0,046	0,045	0,052	0,043	0,041
Colony 1, Replicate 2	0,046	0,047	0,045	0,045	0,047	0,048	0,046	0,064	0,041
Colony 1, Replicate 3	0,044	0,045	0,047	0,044	0,054	0,046	0,045	0,045	0,043
Colony 1, Replicate 4	0,045	0,045	0,045	0,058	0,045	0,046	0,044	0,046	0,046
Colony 2, Replicate 1	0,046	0,047	0,047	0,05	0,046	0,044	0,044	0,044	0,043
Colony 2, Replicate 2	0,046	0,05	0,045	0,045	0,045	0,046	0,05	0,046	0,046
Colony 2, Replicate 3	0,049	0,049	0,046	0,045	0,053	0,046	0,047	0,047	0,044
Colony 2, Replicate 4	0,046	0,051	0,043	0,043	0,044	0,049	0,048	0,05	0,042

Fluorescence intensity after 0h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	30032	29468	47522	30425	37202	30859	38967	39848	40167
Colony 1, Replicate 2	38825	32134	33823	37326	38129	41631	37103	40926	383
Colony 1, Replicate 3	33426	38291	40791	38829	35103	35503	39082	38194	40506
Colony 1, Replicate 4	30451	31664	40586	40661	37774	40046	38716	42854	39830
Colony 2, Replicate 1	31429	32597	33648	34085	40700	31595	28914	36532	40434
Colony 2, Replicate 2	39464	32361	39432	40150	39543	41282	38247	33320	39231
Colony 2, Replicate 3	32329	31485	34366	36754	39980	41296	33348	33391	39612
Colony 2, Replicate 4	31225	36435	37063	38806	42787	40222	36968	36323	38492

Abs600 after 2h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,046	0,045	0,045	0,065	0,047	0,047	0,046	0,044	0,041
Colony 1, Replicate 2	0,047	0,047	0,046	0,048	0,048	0,046	0,047	0,046	0,043
Colony 1, Replicate 3	0,046	0,047	0,046	0,045	0,053	0,047	0,048	0,045	0,044
Colony 1, Replicate 4	0,047	0,047	0,055	0,058	0,047	0,046	0,045	0,046	0,043
Colony 2, Replicate 1	0,078	0,048	0,048	0,049	0,048	0,047	0,046	0,047	0,043
Colony 2, Replicate 2	0,048	0,055	0,048	0,049	0,047	0,046	0,049	0,049	0,041
Colony 2, Replicate 3	0,047	0,048	0,048	0,048	0,047	0,049	0,048	0,05	0,045
Colony 2, Replicate 4	0,046	0,049	0,047	0,046	0,045	0,048	0,048	0,049	0,047

Fluorescence intensity after 2h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	32478	41305	33618	42435	34249	34205	34202	34949	29069
Colony 1, Replicate 2	34345	42706	38570	43860	36547	44267	45267	47187	37138
Colony 1, Replicate 3	38418	39069	38866	44974	36631	43986	46760	39103	39152
Colony 1, Replicate 4	36379	42391	36129	45361	39003	44229	32123	33637	41422
Colony 2, Replicate 1	36339	36658	36464	35639	43270	42121	34385	44627	40854
Colony 2, Replicate 2	40618	43596	36428	44147	44768	43257	45151	46402	39718
Colony 2, Replicate 3	39505	36517	43094	44747	36790	40034	44931	40597	39712
Colony 2, Replicate 4	36751	41857	35023	41187	35201	43423	32927	35063	37786

Abs600 after 4h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,056	0,055	0,055	0,052	0,058	0,052	0,053	0,046	0,042
Colony 1, Replicate 2	0,054	0,056	0,054	0,052	0,055	0,054	0,055	0,048	0,041
Colony 1, Replicate 3	0,055	0,061	0,055	0,052	0,057	0,052	0,055	0,049	0,043
Colony 1, Replicate 4	0,055	0,056	0,05	0,053	0,053	0,052	0,05	0,049	0,043
Colony 2, Replicate 1	0,053	0,059	0,057	0,061	0,053	0,059	0,053	0,059	0,043
Colony 2, Replicate 2	0,054	0,056	0,058	0,062	0,053	0,059	0,055	0,059	0,043
Colony 2, Replicate 3	0,054	0,056	0,058	0,063	0,057	0,059	0,055	0,06	0,043
Colony 2, Replicate 4	0,054	0,056	0,056	0,058	0,049	0,058	0,056	0,061	0,042

Fluorescence intensity after 4h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	39398	41211	43472	34629	40024	37129	34265	40517	38102
Colony 1, Replicate 2	39957	41499	44252	43313	44778	36906	43865	42985	38644
Colony 1, Replicate 3	39198	41382	35548	45574	47239	39866	44132	44431	37943
Colony 1, Replicate 4	35352	41590	26466	44768	45637	44736	29248	37825	38618
Colony 2, Replicate 1	41468	42577	34717	39284	40334	42558	46026	41271	39053
Colony 2, Replicate 2	43892	40143	36619	42028	41878	43940	38303	47567	37978
Colony 2, Replicate 3	43598	43486	44578	44449	44828	44235	42442	37273	39116
Colony 2, Replicate 4	43048	43193	42421	38871	34908	41518	43876	44582	37838

Abs600 after 6h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,098	0,093	0,084	0,084	0,087	0,078	0,083	0,062	0,048
Colony 1, Replicate 2	0,09	0,094	0,082	0,076	0,089	0,083	0,091	0,064	0,041
Colony 1, Replicate 3	0,091	0,09	0,92	0,082	0,089	0,08	0,086	0,066	0,043
Colony 1, Replicate 4	0,086	0,084	0,09	0,079	0,086	0,078	0,084	0,063	0,043
Colony 2, Replicate 1	0,081	0,093	0,094	0,076	0,075	0,097	0,087	0,097	0,043
Colony 2, Replicate 2	0,079	0,092	0,091	0,099	0,073	0,095	0,085	0,107	0,043
Colony 2, Replicate 3	0,08	0,086	0,101	0,13	0,073	0,1	0,088	0,107	0,042
Colony 2, Replicate 4	0,078	0,092	0,089	0,108	0,073	0,093	0,09	0,105	0,042

Fluorescence intensity after 6h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	44819	43197	34990	39952	35312	44026	44108	34395	41891
Colony 1, Replicate 2	35745	41624	42712	36773	38191	39327	36222	38684	44486
Colony 1, Replicate 3	43503	43470	38934	39338	39338	47418	46563	41520	40216
Colony 1, Replicate 4	40336	39507	37827	41517	41517	46271	47662	42298	38353
Colony 2, Replicate 1	34982	34672	46394	18665	18665	36165	36374	40293	40211
Colony 2, Replicate 2	43791	37273	44467	41079	41079	41516	44327	38827	38014
Colony 2, Replicate 3	39248	40869	38169	48933	48933	44903	41210	38643	40437
Colony 2, Replicate 4	38078	38846	41889	49892	49892	42376	46132	38525	39913

19.08.-27.08.17

pH change over time in incubator with RPMI1640 and acid

composition	start pH	incubation time	trend
2ml RPMI1640	7.14	93 min
2ml RPMI1640	7.16	67 min
2ml RPMI1640	7.15	104 min
2ml RPMI1640 + 65µl lactic acid (0.6 M)	6.2	95 min
2ml RPMI1649 + 65µl lactic acid (0.6 M)	6.3	90 min
2ml RPMI1640 + 20µl HCl(1.2 M)	6.6	105 min
2ml RPMI1640 + 30µl HCl(1.2 M)	6.6	100 min
7.5 ml RPMI1640 + 250.000 HEK cells / ml	7.3	24 h
7.5 ml RPMI1640 + 500.000 HEK cells / ml	7.17	26 h

10.08.17

Transformation of pWHE-644

- transformation in DH5-α

12.08.17

Miniprep of pWHE-644 in DH3-α

- miniprep with Zymo kit

Test digest of pWHE-644

1µl NcoI
2µl DNA
1.5µl FD-buffer 10x
10.5µl H20
Excepted bands could be observed.

13.08.17

Miniprep of CMV-VEGFR2 (pIG_138)

- 7x miniprep of CMV-VEGFR2

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl buffer
1µl BamHI
0.5µl H20
Only one of the expected bands could be oberserved.

13.08.17

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl FD-buffer
1µl SmaI
Only one of the expected bands could be oberserved.

16.08.17

pH test with Jurkat cells

- centrifuge Jurkatt cells, dilute in RPMI1640
- count cells
- incubation of Jurkat cells and lactic acid
- pH measurements after 0h, 30min, 1h and 1h30min
- 1) only RPMI1640 with cells, start pH at 7.2
- 2) RPMI1640 with cells and 100µl lactic acid, start pH at 6.8

after 0h:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.46	6.81
2mio	7.41	6.82
1.5mio	7.47	6.86
1mio	7.49	6.79
500k	7.45	6.78
100k	7.45	6.77

after 30min:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.52	7.18
2mio	7.51	7.13
1.5mio	7.53	7.14
1mio	7.52	7.11
500k	7.54	7.07
100k	7.55	7.12

after 1h:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.48	7.26
2mio	7.57	7.22
1.5mio	7.60	7.24
1mio	7.60	7.22
500k	7.60	7.18
100k	7.63	7.21

after 1h30min:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.42	7.14
2mio	7.55	7.27
1.5mio	7.46	7.35
1mio	7.62	7.18
500k	7.51	7.23
100k	7.59	7.26

16.08.17

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl FD-buffer
2µl BamHI
9,5µl H20

Ctla4 induction

- Jurkat cells transiently transfected with Ctla4-GFP-CMV-mCherry
- positive control: cotransfected with CMV-GFP and CMV mCherry
- add VEGF
- incubation for 24h at 37°C

17.08.17

PEI transfection of HEK cells

- CMV-GFP from iGEM
- CMV-GFP from tool box

Flow cytometry

- Jurkat with Ctla4-GFP-CMV-mCherry from 16.08.17

mCherry	GFP (measured in green fluorescence channel)

18.08.17

BioRad electroporation

Cell lines: Jurkat
Plasmids: pIG_037, pIG_031, contransfection CMV-GFP

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 9 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

Calcium test

- incubation of cells at 37°C in FACS tubes for 5min - pre-FACS after 3min - add VEGF (30ng/ml, 70ng/ml) - FACS afer 7min: no signal

20.08.17

Biorad electroporation

Cell lines: Jurkat
Plasmids: pIG_034, cotransfection CMV-GFP and CMV-mCherry, negative control (no DNA)

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 6 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
    

pH tests with Jurkat

V(ges) [ml]	V(RPMI1640) [ml]	V(cells) [ml]	c(cells) [cells/ml]
7.5	5.3	2.2	250.000

- incubation for 24h

Miniprep of pIG_034

- miniprep 8x

21.08.17

sequencing of pIG_138

- plasmid: CMV-VEGFR2 (pIG_138)
- DNA concentration: 80ng/ml
- Oligo: 2µl DNA, 18µl H2O
- V(ges): 20µl

22.08.17

Miniprep of pIG_017, pIG_031, pIG_034, pIG_037

- Zymo kit

up-concentration of pIG_017, pIG_031, pIG_034, pIG_037

- add isopropanol to plasmid suspension in H2O
- -20°C for 20min
- centrifugate for 30min at maximal speed and 4°C
- remove isopropanol
- wash with EtOH (70%)
- centrifugate for 30min att maximal speed and 4°C
- remove EtOH
- open eppis to dry overnight
- eluate with H2O
Up-conentration did not work, DNA concentration decreased.

25.08.17

Miniprep of pIG_017, pIG_037

07.09.17

PEI transfection of HEK cells

used cells: HEK cells

Plasmid number	component
pIG17_034	pCRE-GFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry
pIG17_034 + β2-receptor	pCRE-GFP-CMV-mCherry + β2-receptor
no DNA	negative control

1. Split the cells 1:5 into 10 cm plate
   
2. Thaw PEI reagent at RT
   
3. Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
   
4. Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
   
5. incubate 15 min at RT
   
6. Add the mix to the culture
   
7. incubate for 24 h
   

Gel extraction of M1-receptor without CFP

50 ng DNA
1 µl T4 Ligase
2 µl Buffer 10x
13 µl H2O

08.09.17

Colony PCR of M1

Buffer 10x	2.5µl	12.5 µl
dNTP 10 mM	0.5 µl	2.5 µl
Template	1 µl
Primer	0.5 µl	2.5 µl
DreamTaq Thermo Fisher	0.25 µl	1.25 µl
H2O	20 µl	100 µl

20.08.17

pCRE induction

- treatment in 6-well-plate
- add Forskolin and β2-inductor to the cells

Cells transfected with pIG17_034 (pCRE-GFP-CMV-mCherry):

approach	Forskolin	β2-inductor
negative control	-	-
old Forskolin	10 µM (old)	-
new Forskolin	10 µM (new)	-
new Forskolin	100 µM new Forskolin
β2-inductor	-	10 µM
β2-inductor	-	100 µM

Cells transfected with pIG17_034 (pCRE-GFP-CMV-mCherry) and β2-receptor:

approach	Forskolin	β2-inductor
negative control	-	-
β2-inductor	-	10 µM
β2-inductor	-	100 µM

September

10.09.17

PEI transfection of plasmids pIG17_022/23/37/86

used cells: HEK cells

Plasmid number	component
pIG17_031	HRE-GFP-CMV-mCherry

1. Split the cells 1:2 into 10 cm plate
   
2. Thaw PEI reagent at RT
   
3. Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
   
4. Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
   
5. incubate 15 min at RT
   
6. Add the mix to the culture
   
7. incubate for 24 h
   

HRE induction

- add CoCl2 (end concentrations: 0 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 400 µM, 800 µM)
- fluoresence microscopy

Repetition of the Interlab Study

- transformation of DH5-α with plasmids from Kit 7 (21B, 21D, 21F, 21H, 21L, 21N, 21P)
- incubation at 37 °C

PEI transfection of HEK cells

Plasmid number	component
pIG17_034 + L3 (Luciferase)	pCRE-GFP-CMV-mCherry + L3
pIG17_034 + TDAG8	pCRE-GFP-CMV-mCherry + TDAG8
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry

16.09.17

Pick colonies of Interlab Study

- 2 colonies per plate
- incubation at 37°C

pCRE induction in HEK cells

- plate out cells on 24-well-dish
- treatment with Forskolin

Plasmid number	component	Forskolin treatment
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry	-
pIG17_034 + TDAG8	pCRE-GFP-CMV-mCherry + TDAG8	+
no DNA	-	-
no DNA	-	+
pIG17_034	pCRE-GFP-CMV-mCherry	-
pIG17_034	pCRE-GFP-CMV-mCherry	+

- after 10 hours: pH change to 6.5, 7.0, 7.5
- plate reader after 1h, 3h, 6h, 12h, 24h (Ex.: 488±20 nm; Emm.: 509±20 nm)
- FACS analysis after 24h

FACS

Plate Reader

17.09.17

PEI transfection of plasmid pIG17_031

used cells: HEK cells

Plasmid number	component
pIG17_031	HRE-GFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry

1. Split the cells 1:2 into 10 cm plate (on 16.09.17)
   
2. Thaw PEI reagent at RT
   
3. Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
   
4. Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
   
5. incubate 15 min at RT
   
6. Add the mix to the culture
   
7. incubate for 24 h
   

18.09.17

HRE induction

- induction cancelled because cells were contaminated with bacteria

19.09.17

overnights of overnights (HRE4x, CRE4x)

20.09.17

Midiprep of cultures from overnights

PEI transfection

used cells: HEK cells

Plasmid number	component
+ TDAG8	CRE(4x)-GFP-CMV-mCherry + TDAG8
	HRE(4x)-GFP-CMV-mCherry
pIG17_008 + pIF17_009	CMV-GFP + CMV-mCherry

1. Split the cells 1:2, transfer into 6-well-plate (on 16.09.17)
   
2. Thaw PEI reagent at RT
   
3. Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
   
4. Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
   
5. incubate 15 min at RT
   
6. Add the mix to the culture
   
7. incubate for 24 h
   

Filtration of DMEM, addition of FBS

- filter DMEM sterile
- add 2 % FBS
- set up pH of aliquots with HCl (4 M)
- final pH values: 6.7, 7.1, 7.4

22.09.17

induction of CRE and HRE in HEK cells

- indution in 96-wel-plates - cells transiently transfected with CRE-GFP-CMV-mCherry, add acid
- cells transiently transfected with HRE-GFP-CMV-mCherry, add CoCl2
- plate reader: no measurable induction of GFP

23.09.17

FACS analysis of HEK cells

- FACS analysis after CRE induction and HRE induction

CREx4_GFP	HREx4_GFP

24.09.17

Repetition of the Interlab Study

- transformation of DH5-α with plasmids from Kit 6 (20B, 20D, 20F, 20H, 20L, 20N, 20P)
- incubation at 37 °C

29.09.17

calibration of plate reader

- used cells: knockdown-cells + untransfected cells, compositions 1:0, 1:1, 1:2, 1:4, 0:1
- 96-well-plates, black, clear bottom, 100 000 cells per well
- different media: RPMI164, DMEM, DMEM and PBS (500 000 cells per well)

induction of HRE(4x)

- used cells: HEK stably transfected with HRE(4x)-GFP-CMV-mCherry, untransfected as a negative control
- induction in 24-well-plate
- add CoCl2: 0 µM, 20 µM, 40 µM, 80 µM, 100 µM, 320 µM, 640 µM
- FACS analysis

October

02.10.17

pH measurements with HEK cells

- 6-well-plate, 900 000 cells per well (90% confluency)
- DMEM (5 ml per well), pH set to 6.8
- after 6 h: pH=7.2

induction of HRE

- used cells: Jurkat and HEK, stably transfected with HRE(4x)-GFP-CMV-mCherry
- induction with CoCl2: 0 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM

pH measurements with HEK cells

- 6-well-plate, 900 000 cells per well
- DMEM (5 ml per well), pH set to 6.73

03.10.17

indcution of CRE(4x) in HEK cells

- HEK stably transfected with CRE-GFP-CMV-mCherry, induction with acid (pH 6.7, 7.4, 8.1)
- FACS measurement failed

FACS analysis of CoCl2 treated HEK cells

05.10.17

• resuspended HEK cells containing stable CREx4 were diluted at different ratios with wildtype HEK cells were incubated at different pH values (6.5, 7.1, 7.7) and measured on a plate reader.
• normalization via OD600

14.10.17

Repetition of the Interlab Study

- transformation of positive control, negative control, Device 1-6

15.10.17

pick colonies of the Interlab Study

17.10.17

calibration plate reader

-calibration of plate reader for CFP with dilutions of knockdown Jurkat and HEK cells (CMV_CFP)
-black 96 well plate with clear bottom
-cells/well: 50k, 100k
-wavelengths: Ex.: 433±20 nm; Emm.: 475±9 nm

HEK	Jurkat

cell measurements of the Interlab Study

- OD600 and fluorescence intensity of each device after 0h, 2h, 4h and 6h
- again random fluorescence values

PEI transfection of CMV-VEGFR2, SV40-TDAG8

plasmid
CMV-VEGFR2
SV40-TDAG8

1. Split the cells 1:2 into 10 cm plate
   
2. Thaw PEI reagent at RT
   
3. Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
   
4. Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
   
5. incubate 15 min at RT
   
6. Add the mix to the culture
   
7. incubate for 24 h
   

18.10.17

input tests in HEK and Jurkat cells

used medium: PBS (10% FCS, 0.5% glucose, 1% HEPES)

fluorescence measurement after 0h, 1h, 2h, 4h, 6h, 9h, 16, 24h

in order to determine the relative fluorescence intensity (RFI) stable cell lines were substracted by wildtype cells with same treatment.

CRE(4x)-CFP-CMV-mCherry (stable), SV40-TDAG8 (transient):

conditions:

pH	Forskolin + IBMX (100 µM each)	transient SV40_TDAG8 (HEK only)
6.5	-	-
6.5	+	-
6.5	-	+
6.5	+	+
7.1	-	-
7.1	+	-
7.1	-	+
7.1	+	+
7.7	-	-
7.7	+	-
7.7	-	+
7.7	+	+
6.5 → 7.7	-	-
6.5 → 7.7	+	-
6.5 → 7.7	-	+
6.5 → 7.7	+	+

Results:

HEK293T	Jurkat

Ctla4(4x)-CFP-CMV-mCherry (stable), CMV-VEGFR-2 (transient):

conditions:

VEGF (ng/ml)	Ionomycin (µmol/l)	transient CMV-VEGFR-2 (HEK only)
0		+
0		-
25		+
25		-
50		+
50		-
100		+
100		-
	1.25	+
	1.25	-
	2.5	+
	2.5	-
	5.0	+
	5.0	-
	10.0	+
	10.0	-

Results:

HEK293T	Jurkat

HRE(4x)-CFP-CMV-mCherry (stable):

conditions:

CoCl2 (µmol/ml)
0
50
100
200
400
600

Results:

HEK293T	Jurkat
	high mCherry
	low mCherry

19.10.17

HRE, Ctla4 and HRE induction

- medium change for Ctla4 and HRE cells: PBS (10% FCS, 0.5% glucose, HEPES)
- medium change for CRE: PBS (10% FCS, 0.5% glucose, HEPES) pH = 7.7
- fluorescence measurement after 0h, 3h, 6h, 12h, 24h, 30h