Insulin
<html xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns="http://www.w3.org/TR/REC-html40">
<head> <meta name=Title content=β2-micoglobulin> <meta name=Keywords content=""> <meta http-equiv=Content-Type content="text/html; charset=macintosh"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 11"> <meta name=Originator content="Microsoft Word 11"> <link rel=File-List href="ins_files/filelist.xml"> <title>β2-micoglobulin</title> <!--[if gte mso 9]><xml>
<o:DocumentProperties> <o:Author>Sharmistha Sinha</o:Author> <o:Template>Normal</o:Template> <o:LastAuthor>Sharmistha Sinha</o:LastAuthor> <o:Revision>2</o:Revision> <o:Created>2009-12-12T00:56:00Z</o:Created> <o:LastSaved>2009-12-12T00:56:00Z</o:LastSaved> <o:Pages>1</o:Pages> <o:Words>108</o:Words> <o:Characters>616</o:Characters> <o:Company>Neuro</o:Company> <o:Lines>5</o:Lines> <o:Paragraphs>1</o:Paragraphs> <o:CharactersWithSpaces>756</o:CharactersWithSpaces> <o:Version>11.1282</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings>
</xml><![endif]--><!--[if gte mso 9]><xml>
<w:WordDocument> <w:DisplayHorizontalDrawingGridEvery>0</w:DisplayHorizontalDrawingGridEvery> <w:DisplayVerticalDrawingGridEvery>0</w:DisplayVerticalDrawingGridEvery> <w:UseMarginsForDrawingGridOrigin/> </w:WordDocument>
</xml><![endif]--> <style> <!--
/* Font Definitions */
@font-face {font-family:"Times New Roman"; panose-1:0 2 2 6 3 5 4 5 2 3; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:"Courier New"; panose-1:0 2 7 3 9 2 2 5 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:Wingdings; panose-1:0 5 2 1 2 1 8 4 8 7; mso-font-charset:2; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:0 0 256 0 -2147483648 0;} @font-face {font-family:"\FF2D\FF33 \30B4\30B7\30C3\30AF"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:16777216 0 117702657 0 131072 0;} @font-face {font-family:Verdana; panose-1:0 2 11 6 4 3 5 4 4 2; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:"Arial Narrow"; panose-1:0 2 11 5 6 2 2 2 3 2; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman";} table.MsoNormalTable {mso-style-parent:""; font-size:10.0pt; font-family:"Times New Roman";} table.MsoTableGrid {font-size:10.0pt; font-family:"Times New Roman";} p.MsoNoteLevel1, li.MsoNoteLevel1, div.MsoNoteLevel1 {margin:0in; margin-bottom:.0001pt; text-indent:0in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:1; mso-list:l0 level1 lfo6; tab-stops:list 0in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel2, li.MsoNoteLevel2, div.MsoNoteLevel2 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.75in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:2; mso-list:l0 level2 lfo6; tab-stops:list .5in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel3, li.MsoNoteLevel3, div.MsoNoteLevel3 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:1.25in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:3; mso-list:l0 level3 lfo6; tab-stops:list 1.0in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel4, li.MsoNoteLevel4, div.MsoNoteLevel4 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:1.75in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:4; mso-list:l0 level4 lfo6; tab-stops:list 1.5in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel5, li.MsoNoteLevel5, div.MsoNoteLevel5 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:2.25in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:5; mso-list:l0 level5 lfo6; tab-stops:list 2.0in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel6, li.MsoNoteLevel6, div.MsoNoteLevel6 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:2.75in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:6; mso-list:l0 level6 lfo6; tab-stops:list 2.5in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel7, li.MsoNoteLevel7, div.MsoNoteLevel7 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:3.25in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:7; mso-list:l0 level7 lfo6; tab-stops:list 3.0in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel8, li.MsoNoteLevel8, div.MsoNoteLevel8 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:3.75in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:8; mso-list:l0 level8 lfo6; tab-stops:list 3.5in; font-size:12.0pt; font-family:Verdana;} p.MsoNoteLevel9, li.MsoNoteLevel9, div.MsoNoteLevel9 {margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:4.25in; margin-bottom:.0001pt; text-indent:-.25in; mso-pagination:widow-orphan; page-break-after:avoid; mso-outline-level:9; mso-list:l0 level9 lfo6; tab-stops:list 4.0in; font-size:12.0pt; font-family:Verdana;} @page Section1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.Section1 {page:Section1;}
/* List Definitions */
@list l0 {mso-list-id:-227; mso-list-template-ids:-1249092968;} @list l0:level1 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 1"; mso-level-text:""; mso-level-tab-stop:0in; mso-level-number-position:left; margin-left:0in; text-indent:0in; font-family:Symbol;} @list l0:level2 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 2"; mso-level-text:\F0B7; mso-level-tab-stop:.5in; mso-level-number-position:left; margin-left:.75in; text-indent:-.25in; font-family:Symbol;} @list l0:level3 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 3"; mso-level-text:o; mso-level-tab-stop:1.0in; mso-level-number-position:left; margin-left:1.25in; text-indent:-.25in; font-family:"Courier New";} @list l0:level4 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 4"; mso-level-text:\F0A7; mso-level-tab-stop:1.5in; mso-level-number-position:left; margin-left:1.75in; text-indent:-.25in; font-family:Wingdings;} @list l0:level5 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 5"; mso-level-text:\F0FA; mso-level-tab-stop:2.0in; mso-level-number-position:left; margin-left:2.25in; text-indent:-.25in; font-family:Wingdings;} @list l0:level6 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 6"; mso-level-text:\F0B7; mso-level-tab-stop:2.5in; mso-level-number-position:left; margin-left:2.75in; text-indent:-.25in; font-family:Symbol;} @list l0:level7 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 7"; mso-level-text:o; mso-level-tab-stop:3.0in; mso-level-number-position:left; margin-left:3.25in; text-indent:-.25in; font-family:"Courier New";} @list l0:level8 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 8"; mso-level-text:\F0A7; mso-level-tab-stop:3.5in; mso-level-number-position:left; margin-left:3.75in; text-indent:-.25in; font-family:Wingdings;} @list l0:level9 {mso-level-number-format:bullet; mso-level-style-link:"Note Level 9"; mso-level-text:\F0FA; mso-level-tab-stop:4.0in; mso-level-number-position:left; margin-left:4.25in; text-indent:-.25in; font-family:Wingdings;} @list l1 {mso-list-id:240794998; mso-list-type:hybrid; mso-list-template-ids:677931968 67698689 67698691 67698693 67698689 67698691 67698693 67698689 67698691 67698693;} @list l1:level1 {mso-level-number-format:bullet; mso-level-text:\F0B7; mso-level-tab-stop:.5in; mso-level-number-position:left; text-indent:-.25in; font-family:Symbol;} @list l2 {mso-list-id:827212806; mso-list-type:hybrid; mso-list-template-ids:-1404653784 67698689 67698691 67698693 67698689 67698691 67698693 67698689 67698691 67698693;} @list l2:level1 {mso-level-number-format:bullet; mso-level-text:\F0B7; mso-level-tab-stop:.5in; mso-level-number-position:left; text-indent:-.25in; font-family:Symbol;} @list l3 {mso-list-id:961106908; mso-list-type:hybrid; mso-list-template-ids:314995288 67698689 67698691 67698693 67698689 67698691 67698693 67698689 67698691 67698693;} @list l3:level1 {mso-level-number-format:bullet; mso-level-text:\F0B7; mso-level-tab-stop:.5in; mso-level-number-position:left; text-indent:-.25in; font-family:Symbol;} ol {margin-bottom:0in;} ul {margin-bottom:0in;} --> </style> <!--[if gte mso 9]><xml>
<o:shapedefaults v:ext="edit" spidmax="1027"/>
</xml><![endif]--><!--[if gte mso 9]><xml>
<o:shapelayout v:ext="edit"> <o:idmap v:ext="edit" data="1"/> </o:shapelayout></xml><![endif]-->
</head>
<body bgcolor=white lang=EN-US style='tab-interval:.5in'>
<div class=Section1>
<p class=MsoNormal><b><![if !supportEmptyParas]> <![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 μM<o:p></o:p></p>
<p class=MsoNormal>Cells: Rat Insulinoma Cells<o:p></o:p></p>
<p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p>
<p class=MsoNormal><![if !supportEmptyParas]> <![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]> <![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"'> </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]> <![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]> <![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]> <![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]> <![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"'> </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"'> </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"'>
</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"'> </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"'> </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"'> </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"'> </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"'> </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]> <![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 – <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]> <![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]> <![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"'> </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"'> </span></span><![endif]>Shearman et. Al 1994: PNAS 91: 1470-1474.<o:p></o:p></p>
<p class=MsoNormal><span style="mso-spacerun: yes"> </span><o:p></o:p></p>
<p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p>
<p class=MsoNormal>Cell Plating:<o:p></o:p></p>
<p class=MsoNormal><![if !supportEmptyParas]> <![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]> <![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]> <![endif]><o:p></o:p></p>
<p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p>
<p class=MsoNormal><![if !supportEmptyParas]> <![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 μM in differential media after 24h and add 10 μl 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 μl volume (for 6 replicates). Add 70 μl of 150 μM insulin 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.<o:p></o:p></li>
<li class=MsoNormal style='mso-list:l2 level1 lfo3;tab-stops:list .5in'>The viability of 15μM insulin is ~50% by MTT assay<o:p></o:p></li>
</ul>
<p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p>
</div>
</body>
</html>