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

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Summary

  • Stimulation of 3x 24 wells plates for ELISA

Materials & Methods

Materials

Stock solutions

Conc. Substance Prepared MW (g/mol)
10 mMst-Ht31 27,974 mg / mL DMSO 2797.4
(I prefer 50mM Tris-HCl (pH 7.5))
10 mMSp-6-Bnz -cAMPS5 µmol in 500 µL ddH2O471.4
10 mM8-pCPT -2’-O-Me-cAMP10 µmol in 1000 µL ddH2O507.8
1 mMmPKI
5 mM6-MBC -cAMP450.3

Method

Prepare solutions per plate:

  1. 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)
  2. 60 µL 8-pCPT (10 mM) in 2 mL S0 (300 µM)
  3. 300 µL Sp-6-Bnz (10 mM) in 2 mL S0 (1500 µM)
  4. 300 µL cBIMPS & 300 µL 6-MBC in 2 mL S0 (750 µM each)
  5. 60 µL db-cAMP (50 mM) in 2 mL S0 (1500 µM)
  6. 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)
    1. mPKI (0.4 µM) can be added here as well when used!
  4. Incubate 20 min.
  5. Add 400 μL DMEM S0 to CTR (lane A)
  6. Add 200 μL DMEM S0 to CSE (lane B)
  7. Add 200 μL 8-pCPT or Sp-6-Bnz according to schedule below (lane C, D)
  8. Incubate 25 min.
  9. Prepare 25 mL DMEM S0 with 100% CSE
  10. Dilute 100% CSE to 45% CSE
  11. Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
  12. Incubate 24 h.
1.100.517 S0 S0 S0 st-Ht31
(50 µM)
st-Ht31
(50 µM)
st-Ht31
(50 µM)
123 4 5 6
CTRA
CSEB
CSE+8-p (100 µM)C
CSE+Bnz (500 µM)D
2.100.517 S0 S0 S0 st-Ht31
(50 µM)
st-Ht31
(50 µM)
st-Ht31
(50 µM)
123 4 5 6
CTRA
CSEB
CSE+8-p (100 µM)C
CSE+8-p (100 µM) + mPKI (400 nM)D
2.100.517 S0 S0 S0 st-Ht31
(50 µM)
st-Ht31
(50 µM)
st-Ht31
(50 µM)
123 4 5 6
CTRA
CSEB
CSE+db-cAMP (500 µM)C
CSE+ mPKI (400 nM)D

Notes

  • 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)

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.

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