Calcitonin

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<p class=MsoNormal><span style='font-size:14.0pt'><b><u>Calcitonin<o:p></o:p></u></b></span></p>

<p class=MsoNormal>Protein Concentration: 15&#956;M</p>

<p class=MsoNormal>Cells: PC12 cells</p>

<p class=MsoNormal>Protocol: </p>

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<li class=MsoNormal style='mso-list:l0 level1 lfo3;tab-stops:list .5in'>Incubate
    calcitonin in MiliQ water at a concentration of 1.5 mM for 24 h prior to
    adding to the cells. The protein is very soluble at this concentration. </li>
<li class=MsoNormal style='mso-list:l0 level1 lfo3;tab-stops:list .5in'>Dilute
    this solution to 150 &#956;M in differential media after 24h and add 10
    &#956;l of this solution to the cells. </li>
<li class=MsoNormal style='mso-list:l0 level1 lfo3;tab-stops:list .5in'>Incubate
    for 24h and do the MTT assay.</li>
<li class=MsoNormal style='mso-list:l0 level1 lfo3;tab-stops:list .5in'>If you
    want to do dose dependence with a drug, prepare the dilutions of the drug
    in the media in 70 &#956;l volume (for 6 replicates). Add 70 &#956;l of
    150 &#956;M calcitonin to the drug solution. Take out 10 &#956;l of media
    from the plated cells and add 20 &#956;l of the peptide+ drug to the cells
    (6 replicates). Do MTT/LDH as required.</li>
<li class=MsoNormal style='mso-list:l0 level1 lfo3;tab-stops:list .5in'>The
    viability of 15&#956;M calcitonin is ~70-80% by MTT assay</li>

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