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: 15μM</p>
<p class=MsoNormal>Cells: PC12 cells</p>
<p class=MsoNormal>Protocol: </p>
<ul style='margin-top:0in' type=disc>
<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 μM in differential media after 24h and add 10
μl 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 μl volume (for 6 replicates). Add 70 μl of
150 μM calcitonin to the drug solution. Take out 10 μl of media
from the plated cells and add 20 μl 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 15μM calcitonin is ~70-80% by MTT assay</li>
</ul>
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