User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/16

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Summary

To look at the effect of Protein kinase A (PKA) and Epac on the IL-8 production by airway smooth muscle cells (ASMC's) induced by cigarette smoke extract (CSE), 8-pcpt-2'-o-me-camp (8-pcpt) and 6-Benz-cAMP (6-Benz) are used to activate Epac and PKA respectively. To illustrate the influence of A kinase Anchoring Proteins (AKAP's) an allround inhibitor of PKA (subunit RII) binding to AKAP's HT31 is used. An inactive peptide HT31P is used as an control.

The purpose of the described method is to stimulate the cells with the substances to prepare for future experiments (e.g. ELISA, Western blot)

Materials & Methods

Materials

Stock solutions

  • HT31 (45 μL HT31 + 3 mL DMEM S0) (10 mM)
    • 270 μL HT31
    • 18 mL DMEM S0
  • HT31P (45 μL HT31 + 3 mL DMEM S0)
    • 270 μL HT31P
    • 18 mL DMEM S0
  • 8-pctp (60 μL in 2 mL) (10 mM)
    • 360 μL
    • 12 mL DMEM S0
  • 6-Benz (300 μL in 2 mL) (10 mM)
    • 1800 μL
    • 12 mL DMEM S0
  • CSE 45%
    • 13.5 mL 100% CSE
    • 16.5 mL DMEM S0

Method

  • Remove medium from 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

Related entries

Run 1: Ht31/Ht31P D9/D12


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