|
|
| Line 13: |
Line 13: |
| * New batch of Ht31P to use | | * New batch of Ht31P to use |
| * [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| 16Feb2010]] | | * [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| 16Feb2010]] |
| | *Splitting cells |
|
| |
|
| ==Materials & Methods== | | ==Materials & Methods== |
Revision as of 13:40, 15 March 2010
<html><style type="text/css">
.todo { color: red }
.done { color: green}
</style></html>
 ELISA & hTERT stimulation
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>
|
Summary
- New batch of Ht31P to use
- 16Feb2010
- Splitting cells
Materials & Methods
Materials
Method
- Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S0)
- Rinse twice with warm PBS and remove buffer
- Add 200 μL DMEM S0 with either HT31 or HT31P (see schedule below)
- Add 400 μL DMEM S0 to CTR (lane A)
- Add 200 μL DMEM S0 to CSE (lane B)
- Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D)
- Wait 25 min.
- Prepare 25 mL DMEM S0 with 100% CSE
- Dilute 100% CSE to 45% CSE
- Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
- Incubate 24 h.
Stimulation set-up
|
|
HT31P
1
|
HT31P
2
|
HT31P
3
|
HT31
4
|
HT31
5
|
HT31
6
|
A
(CTR)
|
|
|
|
|
|
|
B
(CSE)
|
|
|
|
|
|
|
C
(CSE+8-pcpt)
|
|
|
|
|
|
|
D
(CSE+6-Benz)
|
|
|
|
|
|
|
CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp
Preparing Sandwich
- Wash coated plate 5x 200 μL PBS
- Remove PBS
- Add 200 μL BSA
- Incubate 1h @ RT and remove BSA
- Dilute samples as shown below for each 24-wells (dilution buffer (μL)/sample (μL))
24-wells (dilution buffer (μL)/sample (μL))
|
|
1
|
2
|
3
|
4
|
5
|
6
|
| A
|
100/100
|
100/100
|
100/100
|
100/100
|
100/100
|
100/100
|
| B
|
150/50
|
150/50
|
150/50
|
175/25
|
175/25
|
175/25
|
| C
|
150/50
|
150/50
|
150/50
|
175/25
|
175/25
|
175/25
|
| D
|
150/50
|
150/50
|
150/50
|
175/25
|
175/25
|
175/25
|
- Wash of BSA with 200 μL Washing buffer 5x
- Add 100 μL of diluted sample
- Incubate ≥1h @ RT
- Wash 5x 200 μL Washing buffer
- Remove buffer
- prepare biotinylated antibody
- Add 100 μL Biotinylated antibody
- Incubate 1h @ RT
- Remove antibody
- Wash 5x 200 μL Washing buffer
- Add 100 μL streptavidin conjugated to HRP
- Incubate 25 min. @ RT
- Remove liquid
- Wash 5x 200 μL Washing buffer
- prepare TBM substrate
- 1.2 μL H2O
- 400 μL TBM stock solution
- 24 mL substrate buffer
- Add 100 μL substrate
- Incubate 30 min. @ RT
- Stop reaction, add 100 μL stopping solution
- Measure absorbance @ 540 nm
- Check duplo standard curve
- Check if values fall within standard curve (preferred)
96-wells plate ELISA
| #
|
01
S0
|
02
S0
|
03
S0
|
04
Ht31
|
05
Ht31
|
06
Ht31
|
07
S0
|
08
S0
|
09
S0
|
10
Ht31
|
11
Ht31
|
12
Ht31
|
A
CTR
|
D12 (5March2010)
|
|
|
|
|
|
D12, P25 (10March2010)
|
|
|
|
|
|
B
CSE
|
|
|
|
|
|
|
|
|
|
|
|
|
C
CSE+8P
|
|
|
|
|
|
|
|
|
|
|
|
|
D
CSE+6Bnz
|
|
|
|
|
|
|
|
|
|
|
|
|
E
CTR
|
D9, P21 (11March2010)
|
|
|
|
|
|
STND 240 pg/mL
|
STND 96 pg/mL
|
STND 38.4 pg/mL
|
STND 15.4 pg/mL
|
STND 6.1 pg/mL
|
STND 2.5 pg/mL
|
F
CSE
|
|
|
|
|
|
|
STND 240 pg/mL
|
STND 96 pg/mL
|
STND 38.4 pg/mL
|
STND 15.4 pg/mL
|
STND 6.1 pg/mL
|
STND 2.5 pg/mL
|
G
CSE+8P
|
|
|
|
|
|
|
STND 1 pg/mL
|
STND 0 pg/mL
|
|
|
|
|
H
CSE+6Bnz
|
|
|
|
|
|
|
STND 1 pg/mL
|
STND 0 pg/mL
|
|
|
BLANCO?
|
BLANCO?
|
Results
Conclusion
Related
Same actions
|
ELISA & hTERT stimulation
