저자는 그녀의 기술 지원을 위해 낸시 모라 감사드립니다. 이 작품은 Consejo Nacional 드 Ciencia Y Tecnologia, 멕시코에서 연구 보조금 48,573-M과 168,098 의해 보조금 IN212308 및 IN205311-2 Direccion에서 일반 드 Asuntos 델 개인 Academico, Universidad Nacional Autonoma 드 멕시코, 멕시코에 의한 자금을 지원했다.

1. 인간의 피에서 호중구의 절연 (PMN) <ol><li> 항응고제로 헤파린 (10 U / ML)로 약 20 ML 인간의 피를 사용하십시오. 피 venopuncture으로 성인 건강 자원 봉사자로부터 수집되었다. UNAM - 모든 실험은 Instituto 드 Investigaciones Biomédicas의 생명 윤리위원회의 승인하에 수행되었다. </li><li> 15 ML 원뿔 원심 분리기 튜브에 PBS에서 6% dextran T500의 2 ML을 넣고 혈액의 10 ML를 추가합니다. 반전 지하철로 두 세 번 혼합하고 적혈구 침강을 할 수 있도록 45 분에 앉아 보자. </li><li> 신선한 15 ML 원뿔 원심 분리기 튜브의 5 ML Ficoll-Paque두고. </li><li> sedimented 적혈구 위에 형성 백혈구 풍부한 플라즈마을 가지고, 신중하게 두 번째 층을 형성 Ficoll-Paque 상단에 피펫. 두 단계가 있는지 확인합니다. </li><li> 4 ° C.에 20 분에 516 XG에 원심 분리기 원심 분리 한 후, 플라즈마 및 Ficoll-Paque의 계면에서 mononuclear 세포의 층이 있습니다. NEutrophils는 튜브의 하단에 있습니다. </li><li> 표면에 뜨는을 제거, 선반 위에 튜브를 활용하여 세포 펠렛을 아프게하고, 감기의 10 ML (4 ° C) PBS 참고 추가하여 세포를 resuspend :. 최대 pipetting하여 세포를 Resuspending이 너무이기 때문에 다운이 권장하지 않습니다를 세포에 손상. </li><li> 4 ° C.에서 5 분 동안 290 XG에서 50 ML 원뿔 원심 분리기 튜브와 원심 분리기에 세포 현탁액을 전송 </li><li> 이전처럼 세포 펠렛을 부숴 10 감기 ML (4 ° C) 적혈구를 lyse 할 수 hypotonic 솔루션 (0.2 % NaCl, 1 % BSA, 20 MM Hepes, 산도 = 7.4).을 추가 정확히 1 분 손으로 부드럽게 관을 회전하여 섞는다. </li><li> 10 감기 ML (4 ° C) hypertonic 솔루션 (1.6 % NaCl, 1 % BSA, 20 MM HEPES, pH를 = 7.4), 믹스와 hemocytometer를 사용하여 셀을 계산합니다 (일반적으로&gt; 95 %는 PMN합니다.)을 추가 참고 : 셀을 계산하는 동안 얼음에 세포 현탁액으로 관을하세요. </li><li> 이전처럼 원심 분리기와 10 PMN을 resuspend추위 7 셀 / ML (4 ° C) PBS. 얼음 계속. 참고 : 호중구는 4에서 가능한 및 기능 유지 8-6에 대한 시간에 대한 ° C. </li></ol> 2. PMN 활성화 <ol><li> 100 PMN의 정지 μl (1 × 10 7 셀 / ML) 1.5 ML Eppendorf 튜브에. 놔 참고 : 샘플 튜브가 최소 중복으로 수행해야합니다. 대개 부정적인 컨트롤은 만 사용 항체, 및 보조 항체를 포함해야합니다. </li><li> 10 μg / ML에서 해당 항 인테그린 또는 안티 FC 수용체 단클론 항체 (적인 mAb)를 추가하고 15 분에 얼음에 품다. </li><li> 한 감기 ML (4 ° C) PBS, microcentrifuge에서 1,743 XG (4,500 RPM)에서 centrifuging하고 표면에 뜨는을 aspirating.을 추가하여 PMN을 씻으십시오 </li><li> 랙에 대한 관의 하단을 클릭하여 세포 펠렛을 부숴, 1 ML 차가운 PBS를 추가하고 두 번 이전 단계에서와 같이을 씻는다. 참고 :이 언 바운드 항체를 제거 씻는다. </li><li> 동일한에 Resuspend의 PMN (에따뜻한의 itial) 볼륨 (37 ° C) PBS는 F의 60 μg / ML (AB &#39;) 2 염소 안티 - 마우스 IgG를 포함하는. 참고 : 보조 안티 마우스 IgG 항체가 첫 번째 반 수용체 항체에 바인딩되며, 수용체의 crosslinking가 발생합니다. </li><li> 관심 핵 요인에 따라 1-20 분에 대해 37 ° C에서 알을 품다. NF-κB 보통 15 분을 위해, 그리고 보통 엘크-1 5 분에. 참고 : 37에서 셀을 잠복기가 4시에 개최 할 수 없습니다 ° C 유도 수용체 crosslinking, ° C. </li><li> 한 ML 차가운 PBS를 추가하고 microcentrifuge에서 1,743 XG에서 3 분 원심 분리기. </li><li> 표면에 뜨는 제거 </li><li> dry-ice/ethanol 욕조에서 즉시 PMN 10 분 동안 거기를 유지 멈춰 봐. </li></ol> 3. 핵의 절연 및 고정 <ol><li> 100 μl 추위에 Resuspend 냉동 PMN 펠릿 (4 ° C) hypotonic 버퍼 (10 MM의 HEPES, 10 MM KCl, 1.5 MM MgCl 2, 1 ㎜ 갓 부가 DL-dithiothreitol [DTT], 산도 = 7.9). 그것은 하나 dry-ice/ethanol 욕조 하나의 튜브를 꺼내하고, hypotonic 버퍼를 추가하기 전에 관의 바닥을 청소 닦아 것이 좋습니다. </li><li> 얼음에 장소, 그리고 trypan 파랑과 핵 정지의 나누어지는 얼룩에 의해 핵의 무결성을 확인합니다. 핵은 원형과 파란색 봐. 그대로 PMN는 물들, 그리고 세포 파편이 불규칙 모양의 푸른 색 입자로 표시되지 않습니다. 참고 : 핵 서스펜션은 많은 손상 셀이나 먼지가 포함 된 경우는 준비를 버리고 다른 샘플과 절차를 반복하는 것이 좋습니다. </li><li> 감기 객실 내 10 분에 microcentrifuge에 775 XG (3,000 RPM)에서 핵을 원심 분리기. 이 포인트 핵에서 매우 약한 및 엑스트라 케어 샘플은 추위와 과도한 힘에 centrifuging하지 유지에서 촬영해야합니다. </li><li> 아주 조심 pipetting하여 표면에 뜨는을 제거합니다. </li><li> 핵 문제를 해결하기 위해 PBS에 찬 4 % paraformaldehyde 100 μl를 추가합니다. 참고 : 펠렛은 매우을 상실합니다D 버퍼를 추가하면 핵을 resuspend에 충분하다. 아래로 Pipetting하며 피해야한다. </li><li> 20 분에 얼음에 품다. </li><li> Immunolabel의 유동 세포 계측법을위한 핵 또는 4에서 최대 24 시간까지 위해를 유지 ° C. </li></ol> 4. 유동 세포 계측법 분석을위한 핵의 Immunolabeling <ol><li> 1 분에 microcentrifuge에서 1,743 XG (4,500 RPM)에서 핵을 원심 분리기, 매우 부드러운 pipetting하여 표면에 뜨는 제거합니다. </li><li> 100 감기 μl (4 ° C) 0.1 % 트리톤 X-100, 핵을 permeabilize하기 위해 PBS에 4 % paraformaldehyde.을 추가 10 분 동안 얼음에 품다. </li><li> 원심 분리기는 microcentrifuge에서 1,743 XG에서 핵을 permeabilized 조심스럽게 표면에 뜨는을 제거합니다. 이 시점에서 핵 알약은 튜브의 하단에 잘 부착하지 않습니다. 이 펠릿을 떨어 도착하면 다시 원심 분리기하는 것이 좋습니다. </li><li> 특이 현상 바인딩 사이트를 차단하고 20 분에 얼음을 품다하기 위해 PBS에 찬 4% 태아 소 혈청 (FBS) 500 μl에 Resuspend 핵. 원심 분리기1743의 핵은 microcentrifuge에서 3 분 동안 XG 조심스럽게 표면에 뜨는을 제거합니다. </li><li> 4퍼센트 FBS와 2.5 μg / 관심의 핵 요소에 대한적인 mAb의 ML을 포함하는 차가운 PBS 100 μl에 핵을 Resuspend. 20 분에 얼음에 품다. 참고 : 대개 부정적인 컨트롤은 안티 핵 인자 항체를 포함 FITC-라벨 차 항체 만합니다. </li><li> 4퍼센트 FBS와 3 분에 1,743 XG에서 centrifuging과 차가운 PBS 500 μl를 두 번 씻으십시오. </li><li> 4퍼센트 FBS 및 10 μg / 해당 FITC-라벨 차 항체의 ML을 포함하는 차가운 PBS 100 μl에, 아주 조심 resuspend 핵을 표면에 뜨는을 제거하고, 20 분에 얼음에 품다. </li><li> 이전 단계에서와 같이 4퍼센트 FBS와 차가운 PBS 500 μl를 두 번 핵을 씻으십시오. </li><li> PBS의 추운 4 % paraformaldehyde 400 μl에 핵을 Resuspend. 참고 : 핵은 샘플이 오염없이 며칠 동안 저장 될 수 있도록 paraformaldehyde에 배치됩니다혈청의 면전에서 발생할 수있는 문제. </li><li> 최대 3 일에 4에 유동 세포 계측법이나 어둠 속에서 매장 ° C에서 즉시 분석 할 수 있습니다. </li></ol> 5. 유동 세포 계측법 분석 <ol><li> 또는 이와 유사한 장치, 흐름 cytometer 같은 FACScan (프랭클린 호수, NJ Becton 디킨슨)에 immunolabeled 핵을 분석합니다. </li><li> 196에서 로그 스케일에서 SSC, 그리고 점 플롯의 게이트 핵, 10-1에서 로그 스케일로 FSC :에 수집 설정을 조정합니다. </li><li> 샘플 당 만 핵을 습득. </li><li> 400 로그 스케일로 설정 FL-1 채널 (506 nm 정도)를 통해 FITC-스테인드 핵의 형광을 분석합니다. </li></ol>

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제작자들은 더 경쟁 금융 이익이 없다는 점 선언합니다.

여기에 설명 된 정화 방법은 짧은 시간에 95 % (현미경 관찰에 의해 평가)보다 큰 결함이있는 unstimulated 호중구의 분리 (PMN)를 할 수 있습니다. 후자가 완전히 lysed되지 않은 경우 때때로 호중구는 적혈구에 의해 오염 될 수 있습니다. 적혈구와 PMN 쉽게 유동 세포 계측법에 의해 서로 다른 세포 집단으로 구별 할 수 있기 때문에 이것은 보통, 기술에 영향을주지 않습니다. 절연 PMN 그런 다음 특정 단클?...

호중구는 말초 혈 1의 가장 풍부한 백혈구 수 있습니다. 염증과 감염 호중구 동안 그들은 방어 2의 첫 번째 줄의 역할을 어디에 해당 사이트에 표시 할 수있는 최초의 세포입니다. 호중구는 phagocytosis, 반응성 산소 종의 생산, degranulation의 lytic 효소의 출시, 그리고 4,5 염증성 크린 시토 킨의 생산 등 여러 가지 항균 메커니즘 3 가지고 있습니다. 호중구는 빠른 속도로 다양한 세포 표면 수용체에서 신호를 통해 작동하게 수명이 짧은 세포입니다. 호중구는 자신의 짧은 수명으로 인해 터미널 셀을 고려하여 염증 과정을 여섯 동안 활성화하지 않는 한 그들은 apoptosis를 받아야하기 때문에, 그것은 그들은 또한 특정 유전자의 전사 수준을 변경하여 자신의 표현형을 수정할 수 있도록 지금 명확되어 있지만. 크린 시토 킨 5 apoptosis 7,8의 억제의 생산은 두있다,mportant 활성화에 의존적 세포 기능은 호중구의 전사 수준에서 조절. 원자력 요소 κB (NF-κB)는 시토 킨 생산 (4)의 전사 조절과 세포 생존과 다양한 세포 유형에 apoptosis 9-11의 규정에 참여하고 있습니다. 핵 인자 활성화로 이어질 신호 경로는 일반적으로 기자 유전자 assays 또는 전기 영동 이동의 assays (EMSA)에 의한 연구하고 있습니다. 호중구는 수명이 짧은 세포이기 때문에 호중구 transfection에 대한 효율적인 기술이 없으므로 다만,이 셀에 transcriptionally 규제 응답의 연구는, 기자 유전자 assays을 수행 할 수 없습니다. EMSA의 assays는 핵 인자 활성화 12,13을 둘러 호중구에 사용 된, 그 방사성 물질의 사용을 포함하기 때문에 그러나,이 방법은 복잡하고 비용이 있습니다. Nucleofection는 successfu 사용되었습니다 또 다른 방법입니다베드로 단핵 세포 14 transfect합니다. 따라서, 적어도 이론적으로는, 핵 인자 정품 인증 (낮은 효율에도 불구하고) transfection에 의해 호중구에서 감지 될 수 있습니다. 그러나,이 기술은 더 비싼 시간이 오래 걸릴뿐 아니라 아마도 덜 정량 것이다. immunostained 세포의 현미경 분석도 핵에 핵 요소를 감지하는 데 사용할 수 있습니다. 사실, 우리는 핵이 방법 15로 NF-κB의 translocation를 발견했습니다. 불행하게도,이 기술은 또한 시간이 많이 걸리는 덜 양적, 그리고 관찰자의 편견에 따라 달라질 수 있습니다. 여기, 우리는 유동 세포 계측법에 의한 분리 및 immunolabeled 핵의 핵 요인의 감지 및 정량화 할 수있는 간단하고 효율적인 방법을 제시한다. 우리는 인간의 말초 혈액에서 호중구를 분리하는 기술을 설명, (F, 안티 - 수용체 항체와 integrins 또는 FC 수용체를 통해 이러한 세포를 자극 분리하고 immunolabel의 핵 및 유동 세포 계측법에 의해 핵을 분석igure 1). 방법이 성공적으로 NF-κB 15 호중구 핵의 엘크-1 16 핵 요소를 검색하는 데 사용되었습니다. 이 방법의 감도는 핵의 핵 요소 수준의 작은 변화를 감지 할 수 있습니다. 이 방법은 다른 세포 유형에서 핵에서 전사 인자의 수준을 분석하는 데 사용할 수 있습니다.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td >Heparin </td>
 <td >PiSA (Mexico)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td >Dextran T500</td>
 <td >Pharmacosmos A/S (Holbaek, Denmark) </td>
 <td>T1-Dextran T500</td>
 <td></td>
 </tr>
 <tr>
 <td >Ficoll-Paque</td>
 <td >Pharmacia</td>
 <td>17-0320-01 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium chloride </td>
 <td >Sigma</td>
 <td>S7653 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate monobasic</td>
 <td >Sigma</td>
 <td>S9638</td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate dibasic</td>
 <td >Sigma</td>
 <td>S9390</td>
 <td></td>
 </tr>
 <tr>
 <td >Bovine serum albumin (BSA)</td>
 <td >Sigma</td>
 <td>A2153</td>
 <td>Cohn Fraction V</td>
 </tr>
 <tr>
 <td >HEPES</td>
 <td >Sigma</td>
 <td>H3375 </td>
 <td></td>
 </tr>
 <tr>
 <td >Potassium chloride</td>
 <td >Sigma</td>
 <td>P9541 </td>
 <td></td>
 </tr>
 <tr>
 <td >Magnesium chloride anhydrous </td>
 <td >Sigma</td>
 <td>M8266 </td>
 <td></td>
 </tr>
 <tr>
 <td >DL-dithiothreitol (DTT) </td>
 <td >Sigma</td>
 <td>D9163</td>
 <td></td>
 </tr>
 <tr>
 <td >Trypan Blue (0.4 % solution)</td>
 <td >Sigma</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td >Paraformaldehyde</td>
 <td >Sigma</td>
 <td>P6148 </td>
 <td></td>
 </tr>
 <tr>
 <td >Triton X-100 </td>
 <td >Sigma</td>
 <td>X100</td>
 <td></td>
 </tr>
 <tr>
 <td >Fetal bovine serum (FBS)</td>
 <td >GIBCO</td>
 <td>10437-028</td>
 <td></td>
 </tr>
 <tr>
 <td >Monoclonal antibody IV.3</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>025-1 </td>
 <td>Human-specific anti-FcRII (CD32) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody 3G8</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>028-2 </td>
 <td>Human-specific anti-FcRIII (CD16) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody TS2/16</td>
 <td >Dana Farber Cancer Research Institute (Boston, MA) </td>
 <td>Donated by Dr. Martin Hemler </td>
 <td>Human-specific anti-&beta;1 integrin (CD29)</td>
 </tr>
 <tr>
 <td >Monoclonal antibody IB4</td>
 <td >University of California, San Francisco </td>
 <td>Donated by Dr. Eric J. Brown </td>
 <td>Human-specific anti-&beta;2 integrin (CD18)</td>
 </tr>
 <tr>
 <td >F(ab')2 goat anti-mouse IgG </td>
 <td >Cappel (Aurora, OH)</td>
 <td>55468</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-mouse IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55522</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-rabbit IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55665</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p50</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-114</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p65</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-109</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>15-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430791</td>
 <td></td>
 </tr>
 <tr>
 <td>50-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430291</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge, Sorvall Tabletop</td>
 <td >Dupont Instruments</td>
 <td>RT 6000D</td>
 <td></td>
 </tr>
 <tr>
 <td>pH-meter</td>
 <td >Corning</td>
 <td>340</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-20</td>
 <td >Gilson </td>
 <td>F123600</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-200</td>
 <td >Gilson </td>
 <td>F123601</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-1000</td>
 <td >Gilson </td>
 <td>F123602</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td >Fisher Scientific</td>
 <td>0267110</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td >Nikon </td>
 <td>Eclipse E600</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td >Nikon </td>
 <td>TMS</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Bath Incubator</td>
 <td >Fisher Scientific </td>
 <td>2IS-M</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5414C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5418</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow Cytometer</td>
 <td >Becton Dickinson (Franklin Lakes, NJ) </td>
 <td>FACScalibur </td>
 <td></td>
 </tr>
 </tbody>
</table>

a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry

호중구는 말초 혈에서 가장 풍부한 백혈구 수 있습니다. 이러한 세포 따라서 미생물을 침해에 대한 방어의 첫 번째 줄되고, 염증 및 감염의 사이트에 게재 처음입니다. 호중구는 phagocytosis, lytic 효소의 출시, 그리고 반응성 산소 종의 생산과 같은 중요한 항균 기능을 가지고 있습니다. 다음과 같은 중요한 방어 기능뿐만 아니라, 호중구는 전 염증성 크린 시토 킨 및 apoptosis의 억제 생산 등의 감염에 대한 응답으로 다른 작업을 수행 할 수 있습니다. 크린 시토 킨은 감염을 선명하게 할 수 다른 백혈구를 모집하고, apoptosis의 억제는 호중구는 감염의 사이트에 더 오래 살 수 있습니다. 이 함수는 스크립트 수준의 규제를받습니다. 호중구는 수명이 짧은 세포이기 때문에 더 effi가 없기 때문에 그러나, 이러한 세포의 transcriptionally 규제 응답의 연구는 종래의 리포터 유전자 방법으로 수행 할 수 없습니다호중구 transfection에 대한 cient 기술. 여기, 우리는 유동 세포 계측법에 의한 분리 및 immunolabeled 핵의 핵 요인의 감지 및 정량화 할 수있는 간단하고 효율적인 방법을 제시한다. 우리는 인간의 말초 혈액에서 순수 호중구를 분리하는 기술을 설명, 안티 - 수용체 항체와 함께이 세포를 자극 분리하고 immunolabel의 핵 및 유동 세포 계측법에 의해 핵을 분석 할 수 있습니다. 방법이 성공적으로 호중구 및 다른 세포 유형에서 핵으로 NF-κB와 엘크-1 핵 요소를 검색하는 데 사용되었습니다. 따라서,이 방법은 세포 유형의 다양한 절연 핵에서 전사 인자의 활성을 분석하기위한 옵션을 나타냅니다.

정화 방법은 여기에 설명하는 것이 95 % (그림 1A)보다 큰 결함으로 unstimulated 호중구 (PMN)를 제공합니다. 절연 PMN 그런 다음 특정 단클론 항체와 crosslinking 특정 수용체​​에 의해 자극 될 수 있습니다. 우리는 FC 수용체와 integrins (그림 1B)를 통해 PMN을 자극했다. 일단 자극, PMN은 lysed하고 핵은 높은 수율로 분리되어 있습니다. 핵은 그러한 특정 항체와 핵 요소 κB (NF-κB), <stro...

Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry at JoVE.com

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

호중구는 혈액에서 가장 풍부한 백혈구 수 있습니다. 호중구는 전 염증성 크린 시토 킨 및 apoptosis의 억제 생산 등 transcriptionally 규제 기능을 가지고 있습니다. 이 기능은 절연 핵에 유동 세포 계측법에 의해 핵 요인의 감지 및 정량화 할 수 있습니다 여기에 제시된 방법으로 공부 할 수 있습니다

유동 세포 계측법에 의해 인간 호중구에 핵 팩터 활성화를 감지 할 간단하고 효율적인 방법

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

a-simple-efficient-method-to-detect-nuclear-factor-activation-human

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JoVE Journal

Immunology and Infection

17.7K 조회수.  University of Alberta. 호중구는 혈액에서 가장 풍부한 백혈구 수 있습니다. 호중구는 전 염증성 크린 시토 킨 및 apoptosis의 억제 생산 등 transcriptionally 규제 기능을 가지고 있습니다. 이 기능은 절연 핵에 유동 세포 계측법에 의해 핵 요인의 감지 및 정량화 할 수 있습니다 여기에 제시된 방법으로 공부 할 수 있습니다

비디오: A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

유동세포계측법에 의해 피해를 입은 척수 조직에서 면역 세포의 양적 평가 : 세포 정제 방법에 대한 소설을 사용

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

연구

면역학 및 감염병학

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

성인 간세포의 혼합 차 문화에서 Macrophages을 분리하는 간단하고 효율적인 방법

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

유동 세포 계측법에 의한 Thymic 긍정적이고 부정적인 선택의 시험

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

의 상호 작용을 연구<em&gt; 황색 포도상 구균</em&gt; 유동 세포 계측법 및 시간 경과 현미경에 의한 호중구와

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

침투 림프구의 분석을위한 비 효소 해리 신선한 인간의 조직에 대한 간단하고 빠른 프로토콜

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

간단하고 효율적인 접근 방법은 돌연변이 백시 니아 바이러스 벡터를 구축하기

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

에 대한 응답으로 인간의 기본 호중구와 단핵구에 의해 항진균 활성의 라이브 영상<em&gt;를했다. 투스</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

전체 혈액에 의해 인간 Monocyte 하위 집합의 Flow Cytometry 분석

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...