Insulin



       &#946;2-micoglobulin

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<![if !supportEmptyParas]> <![endif]>

 Insulin 

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

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

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

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

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

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

<p class=MsoNormal style='text-align:justify'><![if !supportEmptyParas]> <![endif]></o: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'>· <![endif]>RPMI 1640 medium with L-glutamine 500 mL supplemented with;</o:p>

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

<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> <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) </o:p>

<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> <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>

<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 </o:p> <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 <o:p></o:p> <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>

<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> <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>

<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> <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>

<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 <o:p></o:p> <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>

<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) <o:p></o:p> <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 <o:p></o:p> <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> <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> <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> <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>

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

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

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]> <![endif]><o:p></o: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'>· <![endif]>remove the medium;<o:p></o: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'>· <![endif]>add 10 mL of trypsin;<o:p></o: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'>·

<![endif]>wait 03 minutes;<o:p></o: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'>· <![endif]>add 10 mL of RPMI medium;<o:p></o: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'>· <![endif]>centrifuge: 1250 RPMs for 05 minutes;<o:p></o: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'>· <![endif]>remove the medium;<o:p></o: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'>· <![endif]>resuspend the remain pellet in 20 mL of RPMI medium;<o:p></o: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'>· <![endif]>diluted to new cell culture flasks:<o:p></o:p>

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

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

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

<p class=MsoNormal style='text-align:justify;tab-stops:0in'><![if !supportEmptyParas]> <![endif]><o:p></o: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'>· <![endif]>Kapurniotu et. al 1998: EUR J BIOCHEM 251: 208-216;<o:p></o: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'>· <![endif]>Shearman et. Al 1994: PNAS 91: 1470-1474.<o:p></o:p>

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

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

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

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

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

<p class=MsoNoteLevel1 style='mso-list:none;tab-stops:.5in'><span style='font-family:"Times New Roman"'>cell density: 5 x 105 cell| mL = 5 x 104 cells|well;<o:p></o: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>

<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>

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

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

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