User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/18

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Summary

To determine the amount of interleukin 8 (IL-8) produced by the airway smooth muscle cells (ASMC's) ELISA is performed using the PeliKine Compact™ human IL-8 ELISA kit. In this way the effect of PKA, EPAC and CSE on the inflammatory cytokine can be determined.

Materials & Methods

Materials

  • Coating buffer
  • IL-8 antibody Coating antibody
  • 96-wells plate (MaxiSorb)
  • PBS 5%
  • BSA 5% (in PBS)
  • Pelikine Compact™ Human IL-8 ELISA kit
  • Washing buffer (PBS + 0.005% TWEEN 20)
  • IL-8 antibody
  • Streptavidin conjugated to HRP
  • Substrate buffer
    • 1.5 sodiumacetate, resuspend in 80 mL ultrapure water
    • pH 5.5
    • up to 100 mL with ultrapure water
  • TMB stock solution (light sensitive)

Method

Coating of plate

  • Mix
    • 24 mL coating buffer
    • 80 μL primary antibody
  • Add 100 μL per well (96-wells plate)
  • Incubate ON

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 H2O2 (30%)
    • 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 @ 450 nm
    • Check duplo standard curve
    • Check if values fall within standard curve (preferred)

Notes

  • Of the 96-wells plate well F1 contain a Polystyrene fragment

Results

  • Standard curves were accurate (R square 1>0.99)
  • Negative controls showed hardly any IL-8 secretion into the medium (see figures below)


Image 180220101: The average IL-8 secretion of donor 9 corrected for cell viability. Error bars represent standard deviation. CTR: control, CSE: Cigarette smoke extract, 8-p: 8-pcpt-2'-o-me-camp, Bnz:6-Benz-cAMP.

Image 180220102: The average IL-8 secretion of donor 12 corrected for cell viability. Error bars represent standard deviation. CTR: control, CSE: Cigarette smoke extract, 8-p: 8-pcpt-2'-o-me-camp, Bnz:6-Benz-cAMP.

  • Donor 9 showed twice as low IL-8 concentrations (see figures

Conclusion

  • It appears that HT31P affects the cells viability negatively.
  • When comparing the effect of CSE without (HT31P) and with (HT31) PKA/AKAP interaction inhibition, it seems that when AKAP/PKA association is inhibited it decreases CSE induced IL-8 release.
    • PKA/AKAP interaction is required for CSE induced IL-8 secretion.
  • When inducing Epac no effect is seen when comparing the HT31 situation, however slight decrease of IL-8 production is seen when HT31P control situation receives Epac stimulation.
    • When Epac can compete with PKA for the present cAMP it inhibits the increased IL-8 secretion.
    • Epac does not have an influence when PKA can not have its effect
  • Increased PKA activity induces inhibition of IL-8 secretion even without AKAP association
    • It might be that PKA is activated prior to AKAP association, removing its necessity


  • PKA/AKAP induces IL-8 secretion, however an excess of active PKA with/without AKAP association lowers IL-8 secretion
    • Ht31 does not prevent the activation of PKA, only its localization close to cAMP sources and PKA effectors
  • PKA is the more important effector of cAMP compared to Epac

Related entries

Run 1: Ht31/Ht31P D9/D12

Attachment

Excel hTERT D9
Excel hTERT D12


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