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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] ELISA
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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

 * 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)
 * 30 mg TMB
 * 5 mL DMSO

Method
 Coating of plate   Preparing Sandwich   
 * Mix
 * 24 mL coating buffer
 * 80 μL primary antibody
 * Add 100 μL per well (96-wells plate)
 * Incubate ON
 * 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))
 * 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
 * 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
 * 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)
 * Check duplo standard curve
 * Check if values fall within standard curve (preferred)

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

Run 1: Ht31/Ht31P D9/D12

 * Splitting cells 10Feb2010
 * Counting cells 12Feb2010
 * Putting to S0 15Feb2010
 * Stimulating cells 16Feb2010
 * Ending stimulation & Viability 17Feb2010

Attachment
   [[Media:HTERT_Donor_9_ht31_180210.xls| Excel hTERT D9]]    [[Media:HTERT_Donor_12_ht31_180210.xls| Excel hTERT D12]]


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