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FACS - Revision history
2026-05-12T16:03:36Z
Revision history for this page on the wiki
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J at 05:04, 9 March 2008
2008-03-09T05:04:15Z
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J
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WikiSysop at 03:20, 9 March 2008
2008-03-09T03:20:53Z
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WikiSysop
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WikiSysop at 03:20, 9 March 2008
2008-03-09T03:20:35Z
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<a href="http://Opengenome.net/index.php?title=FACS&diff=28864&oldid=28863">Show changes</a>
WikiSysop
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WikiSysop at 03:20, 9 March 2008
2008-03-09T03:20:16Z
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<a href="http://Opengenome.net/index.php?title=FACS&diff=28863&oldid=26139">Show changes</a>
WikiSysop
http://Opengenome.net/index.php?title=FACS&diff=26139&oldid=prev
Ihjung at 11:25, 4 December 2006
2006-12-04T11:25:47Z
<p></p>
<table class="diff diff-contentalign-left" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">Revision as of 11:25, 4 December 2006</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l12" >Line 12:</td>
<td colspan="2" class="diff-lineno">Line 12:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>5. 1ml 의 staining buffer를 첨가하고 부드럽게 mix한다.<br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>5. 1ml 의 staining buffer를 첨가하고 부드럽게 mix한다.<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>6. <del class="diffchange diffchange-inline">우너심하여 </del>(3000rpm, 5분), 상청액을 제거한다.<br /></div></td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>6. <ins class="diffchange diffchange-inline">원심하여 </ins>(3000rpm, 5분), 상청액을 제거한다.<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>7. 50 ul의 2nd Ab를 첨가한다. FITC/PE-conjugated (Fab)'2 타동물 유래의 anti-1차 항체 유래 동물의 항체 Ab (세포가 유래된 동물의 Ig으로 absorbed) 를 사용하면 가장 이상적이다. 그러나 일반적으로 Fluorescence-label된 타동물의 항 1차 항체&nbsp; Ab를 사용하며 이 경우에 1차 항체 적용 단계가 끝난 후 (제 6단계후), 2~10% 의 2차 항체 생성동물의 serum을 이용하여 30분 정도 blocking 단계를 추가한다.<br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>7. 50 ul의 2nd Ab를 첨가한다. FITC/PE-conjugated (Fab)'2 타동물 유래의 anti-1차 항체 유래 동물의 항체 Ab (세포가 유래된 동물의 Ig으로 absorbed) 를 사용하면 가장 이상적이다. 그러나 일반적으로 Fluorescence-label된 타동물의 항 1차 항체&nbsp; Ab를 사용하며 이 경우에 1차 항체 적용 단계가 끝난 후 (제 6단계후), 2~10% 의 2차 항체 생성동물의 serum을 이용하여 30분 정도 blocking 단계를 추가한다.<br /></div></td></tr>
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Ihjung
http://Opengenome.net/index.php?title=FACS&diff=26138&oldid=prev
Ihjung at 11:25, 4 December 2006
2006-12-04T11:25:34Z
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<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">Revision as of 11:25, 4 December 2006</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l58" >Line 58:</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></font><br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></font><br /></div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div><strong><del class="diffchange diffchange-inline">* </del>[[실험에서 사용한 Protocol]] <del class="diffchange diffchange-inline">*</del></strong><br /></div></td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><strong>[[실험에서 사용한 Protocol]] </strong><br /></div></td></tr>
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Ihjung
http://Opengenome.net/index.php?title=FACS&diff=26137&oldid=prev
Ihjung at 11:25, 4 December 2006
2006-12-04T11:25:20Z
<p></p>
<table class="diff diff-contentalign-left" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">Revision as of 11:25, 4 December 2006</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l20" >Line 20:</td>
<td colspan="2" class="diff-lineno">Line 20:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>9. 1ml의 staining buffer를 첨가하고 부드럽게 mix한다.<br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>9. 1ml의 staining buffer를 첨가하고 부드럽게 mix한다.<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>10. <del class="diffchange diffchange-inline">우너심하여 </del>(3000rpm, 5분), 상청액을 제거한다.<br /></div></td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>10. <ins class="diffchange diffchange-inline">원심하여 </ins>(3000rpm, 5분), 상청액을 제거한다.<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>11. 200ul의 staining buffer (PI solution을 첨가한 것 사용) 에 재부유시켜서 즉시 FACS 분석을 실시한다. (PI solution이 세포에 적용된 이후에는 5분 이내에 FACS 분석에 사용하고 늦어도 10분 안에는 FACS 분석을 끝내는 것이 좋다)<br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>11. 200ul의 staining buffer (PI solution을 첨가한 것 사용) 에 재부유시켜서 즉시 FACS 분석을 실시한다. (PI solution이 세포에 적용된 이후에는 5분 이내에 FACS 분석에 사용하고 늦어도 10분 안에는 FACS 분석을 끝내는 것이 좋다)<br /></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></font><br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></font><br /></div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div><strong>* 실험에서 사용한 Protocol *</strong><br /></div></td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><strong>* <ins class="diffchange diffchange-inline">[[</ins>실험에서 사용한 Protocol<ins class="diffchange diffchange-inline">]] </ins>*</strong><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></font></font></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></font></font></div></td></tr>
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Ihjung
http://Opengenome.net/index.php?title=FACS&diff=26136&oldid=prev
Ihjung at 11:24, 4 December 2006
2006-12-04T11:24:46Z
<p></p>
<table class="diff diff-contentalign-left" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">Revision as of 11:24, 4 December 2006</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l55" >Line 55:</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>- Stock solution으로 0.5 mg/ml로 만들어서 냉동보관 (-20도)<br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>- Stock solution으로 0.5 mg/ml로 만들어서 냉동보관 (-20도)<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>- staining buffer에 1/200으로 희석하여 사용<br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>- staining buffer에 1/200으로 희석하여 사용<br /></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"><br /></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"><strong>* 실험에서 사용한 Protocol *</strong><br /></ins></div></td></tr>
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Ihjung
http://Opengenome.net/index.php?title=FACS&diff=25955&oldid=prev
Ihjung at 12:52, 22 November 2006
2006-11-22T12:52:27Z
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<td colspan="2" style="background-color: #fff; color: #222; text-align: center;">Revision as of 12:52, 22 November 2006</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l25" >Line 25:</td>
<td colspan="2" class="diff-lineno">Line 25:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>12. FL3에 양성인 세포는 gate-out 시킨다. (PI에 염색된 세포로 죽은 세포들임)<br /></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>12. FL3에 양성인 세포는 gate-out 시킨다. (PI에 염색된 세포로 죽은 세포들임)<br /></div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div></font></div></td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><font style="BACKGROUND-COLOR: #ccffcc" size="4"><strong>Intracellular stain 법<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"></strong><font style="BACKGROUND-COLOR: #ffffff" size="2">위의 protocol을 사용하는데, step 1 후에 다음의 과정을 추가한다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">(intracellular stain을 위한 kit를 구입하여 실시하면 더욱 간편하다.)<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">1) Cell을 고정시킨다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">&nbsp;- 고정액은, ethanol, acetone, 1% Paraform aldehyde, 1% formalin 등을 사용할 수 있다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">&nbsp;- 10분 정도 실온에서 고정한 후에, 충분한 양의 FACS buffer로 wash해 준다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">2) 고정된 세포막에 항체가 통과할 수 있는 pore를 만들기 위한 처리를 해준다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">&nbsp;- 일반적으로 (0.1% saponin, 0.5% BSA, 0.02% NaN3 in PBS) 액을 사용한다 (BSA나 NaN3는 생략가능)<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">&nbsp;- 실온에서 약 20분 배양한다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">3) 위의 protocol에서 step2 이후를 동일하게 실시한다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">필요한 시약 및 참고사항<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">-&nbsp;항체 : 항체는 대체로 100~500ug.ml의 농도로 공급되며 필요한 양 만큼만을 따다가 희석해 놓는다. (10X로 희석), 즉 500ug/ml의 원액항체의 경우 300ul의 10X 항체액을 만들 경우, 30ul의 항체원액을 따다가 270ul의 항체 희석액을 섞어서 만든다. (원액항체는 분주하지 않고 보관함.) 희석한 항체는 4도씨에서 보관한다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">(새로운 항체를 이용할 때는 항체를 계단 희석하여 가장 최적의 항체농도를 결정한 후에 사용해야 한다. 그러나 일반적으로 1~5ug/ml의 농도가 적당하다)<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">- 항체 희석액 : PBS containing 0.02% sodium azide (NaN3), 0.1% BSA<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">- FACS buffer : PBS containing 0.01% sodium azide, 2~5% serum (염색하고자 하는 세포가 유래한 동물종의 serum) (serum을 넣는 것은 Fc receptor의 blocking 용이므로 Fc receptor가 없는 세포의 경우에는 안넣어도 됨). high background sample의 경우에 세포가 유래한 동물의 serum을 2~10% 첨가하면 background stain을 낮출 수 있다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">참고) Blocking을 위해서 2) 단계 다음에, 항체를 만든 동물의 serum 2~5%를 첨가한 PBS를 100ul 넣어주고 30분간 배양하여 우너심으로 상청액을 제거하고 90ul의 FACS buffer에 재부유시켜 3)의 단계로 진행하기도 한다.<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">Propidium Iodide (PI)<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">- Stock solution으로 0.5 mg/ml로 만들어서 냉동보관 (-20도)<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">- staining buffer에 1/200으로 희석하여 사용<br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"></font><br /></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color: #222; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"></font></ins></font></div></td></tr>
</table>
Ihjung
http://Opengenome.net/index.php?title=FACS&diff=25887&oldid=prev
Ihjung at 11:52, 21 November 2006
2006-11-21T11:52:12Z
<p></p>
<p><b>New page</b></p><div><strong><font style="BACKGROUND-COLOR: #ccffcc" size="4">1 - color FACS Staining Procedure</font></strong><br /><br />
<br /><br />
<br /><br />
<font size="2">1. Cell을 FACS buffer에 부유시킴&nbsp;(약 10의 5제곱 cell / 50 ul 정도 되도록 함) 부착세포의 경우에는 trypsin 또는 EDTA 등을 활용하여 부유시킨다. 이때 trypsin 등의 처리에 의해 표적의 표면 분자가 영향을 받는 경우에는 기타 방법을 고려하여야 한다.<br /><br />
<br /><br />
2. 50 ul을 tube에 분주한다.<br /><br />
<br /><br />
3. 50 ul의 항체를 첨가한다. 일반적으로 5 ug / ml 의 항체 농도가 되도록 항체를 첨가한다. 그러나, 적용하는 최적의 항체 농도를 결정하기 위해서는 각각의 항체별로 titration을 하여 적정한 항체 농도를 결정하여야 한다.<br /><br />
<br /><br />
4. 냉장 또는 on ice 상테에서 1시간 배양한다. (경우에 따라서는 30분간 하여도 무방하다.)<br /><br />
<br /><br />
5. 1ml 의 staining buffer를 첨가하고 부드럽게 mix한다.<br /><br />
<br /><br />
6. 우너심하여 (3000rpm, 5분), 상청액을 제거한다.<br /><br />
<br /><br />
7. 50 ul의 2nd Ab를 첨가한다. FITC/PE-conjugated (Fab)'2 타동물 유래의 anti-1차 항체 유래 동물의 항체 Ab (세포가 유래된 동물의 Ig으로 absorbed) 를 사용하면 가장 이상적이다. 그러나 일반적으로 Fluorescence-label된 타동물의 항 1차 항체&nbsp; Ab를 사용하며 이 경우에 1차 항체 적용 단계가 끝난 후 (제 6단계후), 2~10% 의 2차 항체 생성동물의 serum을 이용하여 30분 정도 blocking 단계를 추가한다.<br /><br />
<br /><br />
8. 냉장 또는 on ice 상태에서 1시간 배영한다 (경우에 따라서는 30분간 하여도 무방하다)<br /><br />
<br /><br />
9. 1ml의 staining buffer를 첨가하고 부드럽게 mix한다.<br /><br />
<br /><br />
10. 우너심하여 (3000rpm, 5분), 상청액을 제거한다.<br /><br />
<br /><br />
11. 200ul의 staining buffer (PI solution을 첨가한 것 사용) 에 재부유시켜서 즉시 FACS 분석을 실시한다. (PI solution이 세포에 적용된 이후에는 5분 이내에 FACS 분석에 사용하고 늦어도 10분 안에는 FACS 분석을 끝내는 것이 좋다)<br /><br />
<br /><br />
12. FL3에 양성인 세포는 gate-out 시킨다. (PI에 염색된 세포로 죽은 세포들임)<br /><br />
</font></div>
Ihjung