User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/16

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ELISA & hTERT stimulation <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Summary

Materials & Methods

Materials


Method

Stimulation hTERT cells

  • 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)
    • Incubate 20 min.
  • 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

ELISA

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
    • It will turn yellow
  • 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 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010)
B
CSE 		D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010)
C
CSE+8P 		D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010)
D
CSE+6Bnz 		D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12 (5March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010) D12, P25 (10March2010)
E
CTR 		D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) 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 		D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) 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
G
CSE+8P 		D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) STND 1 pg/mL STND 0 pg/mL
H
CSE+6Bnz 		D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) D9, P21 (11March2010) STND 1 pg/mL STND 0 pg/mL

Results

  • ELISA IL-8

Conclusion

  • DONE Ow..

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