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

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


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Summary

 * Stimulation of 3x 24 wells plates for ELISA

Materials
Stock solutions

Prepare solutions per plate:

 * 1) 45 µL st-Ht31 (10 mM) in 3 mL S0 (150 µM)
 * 2) 1000 µL of 150 µM st-Ht31 + 1.20 µL of mPKI (1.2 µM)
 * 3) 60 µL 8-pCPT (10 mM) in 2 mL S0 (300 µM)
 * 4) 300 µL Sp-6-Bnz (10 mM) in 2 mL S0 (1500 µM)
 * 5) 300 µL cBIMPS & 300 µL 6-MBC in 2 mL S0 (750 µM each)
 * 6) 60 µL db-cAMP (50 mM) in 2 mL S0 (1500 µM)
 * 7) 4.5 mL 100% CSE + 5.5 mL S0 (45% CSE) (NOT IN ADVANCE)

Procedure

 * 1) Remove medium from 24-wells plate with ASMC's grown ON in DMEM (S0)
 * 2) Rinse twice with warm PBS and remove buffer
 * 3) Add 200 μL DMEM S0 with or without st-Ht31 (see schedule below)
 * 4) mPKI (0.4 µM) can be added here as well when used!
 * 5) Incubate 20 min.
 * 6) Add 400 μL DMEM S0 to CTR (lane A)
 * 7) Add 200 μL DMEM S0 to CSE (lane B)
 * 8) Add 200 μL 8-pCPT or Sp-6-Bnz according to schedule below (lane C, D)
 * 9) Incubate 25 min.
 * 10) Prepare 25 mL DMEM S0 with 100% CSE
 * 11) Dilute 100% CSE to 45% CSE
 * 12) Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
 * 13) Incubate 24 h.

Results

 * See ELISA (17May2010)

Discussion

 * 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.

Related topics

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