User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/17

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Cell stimulation

Summary

Stock solutions

45 µL st-Ht31 (10 mM) in 3 mL S0 (150 µM)
1. 1000 µL of 150 µM st-Ht31 + 1.20 µL of mPKI (1.2 µM)
60 µL 8-pCPT (10 mM) in 2 mL S0 (300 µM)
300 µL Sp-6-Bnz (10 mM) in 2 mL S0 (1500 µM)
300 µL cBIMPS & 300 µL 6-MBC in 2 mL S0 (750 µM each)
60 µL db-cAMP (50 mM) in 2 mL S0 (1500 µM)
4.5 mL 100% CSE + 5.5 mL S0 (45% CSE) (NOT IN ADVANCE)

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 or without st-Ht31 (see schedule below)
1. mPKI (0.4 µM) can be added here as well when used!
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-pCPT or Sp-6-Bnz according to schedule below (lane C, D)
Incubate 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.

All prepared solutions were made together for all plates
The st-Ht31 solution was prepared in DMSO, this caused that solubilization required vortexing and heating of the medium. It looked like addition of glycerol (gel-like) with small insoluble fragment still remaining.
mPKI solution was prepared and added with the st-Ht31
st-Ht31(/mPKI) incubation was prolonged (11.46 - 12.26) to ~40 min.
Used 5 mM 8-pCPT (240 μL)

In Groningen st-Ht31 is purchased from Promega (in Berlin is homemade), Promega suspends it in 50mM Tris-HCl (pH 7.5) which I would prefer for future use. Also because DMSO is toxic for cells, which might interfere with the experiment.