Bitan:Cell Culture
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#EDFFEB;border-collapse:collapse;border:none;mso-border-alt:dashed #CCFFCC 3.0pt; mso-padding-alt:0in 5.4pt 0in 5.4pt'> <tr> <td width=868 valign=top style='width:12.05in;border:dashed #CCFFCC 3.0pt; padding:0in 5.4pt 0in 5.4pt'> <p class=MsoNormal align=center style='text-align:center'><span style='font-size:16.0pt;color:#993300'><b>Basic methods for culturing PC12 cells<o:p></o:p></b></span></p> <p class=MsoNormal><span style='color:#993300'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal>PC12 is a cell line derived from a pheochromocytoma of the rat adrenal medulla.</p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p> <p class=MsoNormal><span style='font-size:14.0pt'><b>Growth media formulation<o:p></o:p></b></span></p> <p class=MsoNormal>Ham’s F12K media (2 mM glutamine) + 15% Horse Serum (heat inactived) + 2.5% Fetal Bovine Serum + 1% Antibiotic-Antimycotic (100X)</p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p> <p class=MsoNormal><span style='font-size:14.0pt'><b>Differentiation media formulation<o:p></o:p></b></span></p> <p class=MsoNormal>Ham’s F12K media (2 mM glutamine) + 0.5% Fetal Bovine Serum + 100 ng/mL NGF + 1% Antibiotic-Antimycotic (100X)</p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p> <p class=MsoNormal><span style='font-size:14.0pt'><b>Heat inactivation of Horse serum<o:p></o:p></b></span></p> <p class=MsoNormal>1. Remove horse serum from -80°C.</p> <p class=MsoNormal>2. Sit in room temperature for 15 min and then thaw at 37°C.</p> <p class=MsoNormal>3. When serum is thawed, place serum in 50°C water bath for 10 min (it is very important that it is only 10 min.)</p> <p class=MsoNormal>4. Remove from water bath and make up media.</p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p> <p class=MsoNormal><span style='font-size:14.0pt'><b>Starting cells<o:p></o:p></b></span></p> <p class=MsoNormal>1. Warm growth media in 37°C water bath for about 20 min.</p> <p class=MsoNormal>2. Take two 75cm<sup>2</sup> flasks. Pipet 10 ml of growth media into each culture flask. Lay horizontally into the incubator for 15 min to adjust pH.</p> <p class=MsoNormal>3. After 15 min, place the flasks into hood.</p> <p class=MsoNormal>4. Take the frozen cells from liquid nitrogen tank.</p> <p class=MsoNormal>5. Thaw cells in 37°C water bath in 1 min.</p> <p class=MsoNormal>6. Pipet cells into 2 flasks. </p> <p class=MsoNormal>7. Place the cells back into incubator. </p> <p class=MsoNormal>8. Change media the second day.</p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p> <p class=MsoNormal><span style='font-size:14.0pt'><b>Subculturing<o:p></o:p></b></span></p> <p class=MsoNormal>1. Warm growth media in 37°C water bath for about 20 min.</p> <p class=MsoNormal>2. Remove cells from incubator and check under microscope.</p> <p class=MsoNormal>3. Remove culture media by aspiration with a glass pipette. Be careful. Don’t disturbing the cells.</p> <p class=MsoNormal>4. Add 6 mL of media to each flask and rinse cells from flask by gently aspirating and dispensing media over the cells (Split 1 to 2-3 depending).</p> <p class=MsoNormal>5. Add 2 mL or 3 mL of cells to new flasks with 8 mL of media.</p> <p class=MsoNormal>6. Return flasks to incubator. </p> <p class=MsoNormal>7. Clean hood with ethanol and wipe with kimwipes.</p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p> <p class=MsoNormal><span style='font-size:14.0pt'><b>Freezing cells<o:p></o:p></b></span></p> <p class=MsoNormal>1. Thaw freezing medium in 4°C and keep in 4°C until use.</p> <p class=MsoNormal>2. Remove cell flasks form incubator. Remove culture media by aspiration with a glass pipette. </p> <p class=MsoNormal>3. Collect cells with fresh pre-warmed growth media.</p> <p class=MsoNormal>4. Count cells.</p> <p class=MsoNormal>5. Calculate the volume of freezing medium needed to cell density 1×10<sup>7</sup>~5×10<sup>6</sup> cells/mL.</p> <p class=MsoNormal>6. Centrifuge cells at 200 g for 5-10 min. Remove media by aspiration with glass pipette. </p> <p class=MsoNormal>7. Resuspend cells in 4°C calculated amount of freezing medium. </p> <p class=MsoNormal>8. Fill 1 mL cells to each cryovial.</p> <p class=MsoNormal>9. Transfer cryovials to freezing box.<span style="mso-spacerun: yes"> </span>The freezing box filled with 100% isopropylalcohol to the marked line. </p> <p class=MsoNormal>10. Sit the freezing chamber in -80°C for 24 hours and then transfer to liquid nitrogen to store.</p> <p class=MsoNormal><span style="mso-spacerun: yes"> </span></p> <p class=MsoNormal>The method is modified from Dr. Erica A. Fradinger’s protocol.</p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><o:p></o:p></p> <p class=MsoNormal><span style='color:#231F20'>More information about PC12 at: <a href="http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1721&Template=cellBiology">http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1721&Template=cellBiology</a></span><span style='font-size:16.0pt'><b><o:p></o:p></b></span></p> </td> </tr>
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