Insulin

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<div class=Section1>

<p class=MsoNormal><b><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></b></p>

<p class=MsoNormal><b><u>Insulin<o:p></o:p></u></b></p>

<p class=MsoNormal>Protein Concentration: 5 &#956;M<o:p></o:p></p>

<p class=MsoNormal>Cells: Rat Insulinoma Cells<o:p></o:p></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal>Cell Culture Protocol:<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify'>TO PREPARE THE MEDIUM:<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>RPMI 1640 medium with L-glutamine 500 mL supplemented with;<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<table border=1 cellspacing=0 cellpadding=0 style='border-collapse:collapse;

border:none;mso-border-top-alt:2.25pt;mso-border-left-alt:.5pt;mso-border-bottom-alt:
2.25pt;mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:
solid;mso-padding-alt:0in 5.4pt 0in 5.4pt'>
<tr>
 <td width=257 valign=top style='width:257.4pt;border-top:2.25pt;border-left:
 .5pt;border-bottom:2.25pt;border-right:.5pt;border-color:windowtext;
 border-style:solid;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'><![if !supportEmptyParas]>&nbsp;<![endif]><span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=131 valign=top style='width:130.5pt;border-top:solid windowtext 2.25pt;
 border-left:none;border-bottom:solid windowtext 2.25pt;border-right:solid windowtext .5pt;
 mso-border-left-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>FINAL
 CONCENTRATION<span style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=55 valign=top style='width:54.9pt;border-top:solid windowtext 2.25pt;
 border-left:none;border-bottom:solid windowtext 2.25pt;border-right:solid windowtext .5pt;
 mso-border-left-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>VOLUME<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
</tr>
<tr>
 <td width=257 valign=top style='width:257.4pt;border:solid windowtext .5pt;
 border-top:none;mso-border-top-alt:solid windowtext 2.25pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>heat-inactivated
 fetal bovine serum (FBS)<span style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=131 valign=top style='width:130.5pt;border-top:none;border-left:
 none;border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext 2.25pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>10%<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=55 valign=top style='width:54.9pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext 2.25pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>50 mL<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
</tr>
<tr>
 <td width=257 valign=top style='width:257.4pt;border:solid windowtext .5pt;
 border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>penicillin|
 streptomycin<span style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=131 valign=top style='width:130.5pt;border-top:none;border-left:
 none;border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>0.1
 mg | mL<span style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=55 valign=top style='width:54.9pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>5 mL<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
</tr>
<tr>
 <td width=257 valign=top style='width:257.4pt;border:solid windowtext .5pt;
 border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>L-glutamine<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=131 valign=top style='width:130.5pt;border-top:none;border-left:
 none;border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>2 mM<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=55 valign=top style='width:54.9pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>5 mL<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
</tr>
<tr>
 <td width=257 valign=top style='width:257.4pt;border:solid windowtext .5pt;
 border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>D-glucose<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=131 valign=top style='width:130.5pt;border-top:none;border-left:
 none;border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>1 mg
 | mL<span style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=55 valign=top style='width:54.9pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>5 mL<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
</tr>
<tr>
 <td width=257 valign=top style='width:257.4pt;border:solid windowtext .5pt;
 border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>non
 essential amino acids (NEAA)<span style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=131 valign=top style='width:130.5pt;border-top:none;border-left:
 none;border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>0.1
 mM<span style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=55 valign=top style='width:54.9pt;border-top:none;border-left:none;
 border-bottom:solid windowtext .5pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>5 mL<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
</tr>
<tr>
 <td width=257 valign=top style='width:257.4pt;border-top:none;border-left:
 solid windowtext .5pt;border-bottom:solid windowtext 2.25pt;border-right:
 solid windowtext .5pt;mso-border-top-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>Na-pyruvat<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=131 valign=top style='width:130.5pt;border-top:none;border-left:
 none;border-bottom:solid windowtext 2.25pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>1 mM<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
 <td width=55 valign=top style='width:54.9pt;border-top:none;border-left:none;
 border-bottom:solid windowtext 2.25pt;border-right:solid windowtext .5pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center;tab-stops:0in'>5 mL<span
 style='font-family:"Arial Narrow"'><o:p></o:p></span></p>
 </td>
</tr>

</table>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'>TO SPLIT THE CELLS:<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>remove the medium;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>add 10 mL of trypsin;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

</span></span><![endif]>wait 03 minutes;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>add 10 mL of RPMI medium;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>centrifuge: 1250 RPMs for 05 minutes;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>remove the medium;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>resuspend the remain pellet in 20 mL of RPMI medium;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l3 level1 lfo4;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>diluted to new cell culture flasks:<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'>1:20 dilution: 19 ml medium + 01 mL culture<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'>1:40 dilution: 19.5 ml medium + 0.5 mL culture<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'>1:3 dilution: 13 ml medium + 07 mL culture &#8211; <b>when experiment is planned!</b><span style='font-weight:normal'><o:p></o:p></span></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'>References:<o:p></o:p></p>

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l1 level1 lfo5;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>Kapurniotu et. al 1998: EUR J BIOCHEM 251: 208-216;<o:p></o:p></p>

<p class=MsoNormal style='margin-left:0in;text-align:justify;text-indent:0in; mso-list:l1 level1 lfo5;tab-stops:list 0in'><![if !supportLists]><span style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>Shearman et. Al 1994: PNAS 91: 1470-1474.<o:p></o:p></p>

<p class=MsoNormal><span style="mso-spacerun: yes">&nbsp;</span><o:p></o:p></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal>Cell Plating:<o:p></o:p></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNoteLevel1 style='mso-list:none;tab-stops:.5in'><span style='font-family:"Times New Roman"'>plate for MTT:<o:p></o:p></span></p>

<p class=MsoNoteLevel1 style='mso-list:none;tab-stops:.5in'><span style='font-family:"Times New Roman"'>day 01:<o:p></o:p></span></p>

<p class=MsoNoteLevel1 style='mso-list:none;tab-stops:.5in'><span style='font-family:"Times New Roman"'>cell density: 5 x 10<sup>5</sup> cell| mL = 5 x 10<sup>4</sup> cells|well;<o:p></o:p></span></p>

<p class=MsoNoteLevel1 style='margin-left:0in;text-indent:0in'><span style='font-family:"Times New Roman"'>add 100 uLs in each well;<o:p></o:p></span></p>

<p class=MsoNoteLevel1 style='margin-left:0in;text-indent:0in'><span style='font-family:"Times New Roman"'>incubate for 24 hrs at incubator;<o:p></o:p></span></p>

<p class=MsoNoteLevel1 style='margin-left:0in;text-indent:0in'><span style='font-family:"Times New Roman"'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>

<p class=MsoNoteLevel1 style='margin-left:0in;text-indent:0in'><span style='font-family:"Times New Roman"'>DAY 02:<o:p></o:p></span></p>

<p class=MsoNoteLevel1 style='margin-left:0in;text-indent:0in'><span style='font-family:"Times New Roman"'>CHANGE THE RPMI MEDIA FOR A FRESH ONE WITH 1% FBS<o:p></o:p></span></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

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

<ul style='margin-top:0in' type=disc>

<li class=MsoNormal style='mso-list:l2 level1 lfo3;tab-stops:list .5in'>Incubate
    insulin in 10mM NaAc (pH 3.2) at a concentration of 2 mM for 24 h prior to
    adding to the cells. The protein is very soluble at this concentration. <o:p></o:p></li>
<li class=MsoNormal style='mso-list:l2 level1 lfo3;tab-stops:list .5in'>Dilute
    this solution to 50 &#956;M in differential media after 24h and add 10
    &#956;l of this solution to the cells. <o:p></o:p></li>
<li class=MsoNormal style='mso-list:l2 level1 lfo3;tab-stops:list .5in'>Incubate
    for 24h and do the MTT assay.<o:p></o:p></li>
<li class=MsoNormal style='mso-list:l2 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 insulin 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.<o:p></o:p></li>
<li class=MsoNormal style='mso-list:l2 level1 lfo3;tab-stops:list .5in'>The
    viability of 15&#956;M insulin is ~50% by MTT assay<o:p></o:p></li>

</ul>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

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