User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/17

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Owwnotebook icon.pngHt31P toxicity <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Summary

  • Stop stimulation
  • Prepare washing buffer 1x ELISA + 10x PBS
  • CoIP

Materials & Methods

Method

Preparations for ELISA & Viability staining

Co-immunoprecipitation Part III

  • Centrifuge sample incubated with beads, 5 min. 14.000 RPM
  • Remove supernatant* (w. syringe)
  • Add 1 mL PBS
  • Centrifuge, 5 min. 14.000 RPM
  • Remove supernatant*
  • Add 75 μL Laemli buffer to beads
  • Store samples @ -20 °C


* Be careful not to touch beads

Results

  • PICTURES OF CELL DEATH (Anouk)
  • Viability staining
Ht31P (μM) Ht31P (μM) Ht31P (μM) Ht31P (μM) Ht31P (μM) Ht31P (μM)
D9 P23 0 10 20 30 40 50
CTR 7524 4117 2092 1929 1346 1712
CTR 6719 3030 1540 1323 1126 1458
CSE 3996 922 649 547 527 660
CSE 3509 1125 646 608 550 577

SurvivalRateD9P2317032010.JPG

Ht31P (μM) Ht31P (μM) Ht31P (μM) Ht31P (μM) Ht31P (μM) Ht31P (μM)
D12 P27 0 10 20 30 40 50
CTR 7151 5242 3640 2953 2435 2331
CTR 6816 4353 2746 2339 1970 2074
CSE 5284 1784 1234 939 938 918
CSE 4858 1891 1250 1020 870 907

SurvivalRateD12P2717032010.JPG

Conclusion

  • Ht31P is lethal for airway smooth muscle hTERT cells
  • Strength of effect is donor dependent