Name: neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents
Uploaded: 2016-03-25T15:00:00.000+00:00
Duration: 14 min 4 s
Description: Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

기본 백혈병 세포 1. 호중구의 분리 및 공동 문화 참고 : 절차는 동의서에 서명 모든 환자와 리옹 병원 윤리위원회의 승인하에 실시 하였다. <ol><li> 기본 백혈병 세포 및 호중구의 분리 <ol><li> 만성 림프 구성 백혈병 (CLL)으로 진단 된 환자에서 EDTA (혈액의 밀리리터 당 1.8 mg을 EDTA)에 말초 혈액의 튜브를 수집합니다. </li><li> 멸균 50 ㎖ 튜브에 혈액을 각각 15 ㎖에 첨가하고 15 ml의 RPMI로 희석 하였다 (희석 1 : 1)로 조심스럽게 천천히 위상 혼합없이 상기 튜브의 하단에 밀도 구배 용액의 15 mL로 추가한다. 밀도 구배 용액을 제조 RT에있을 수 있도록한다. </li><li> RT에서 브레이크없이 35 분 동안 300 XG에 원심 분리기. 그림 1A, 위에서 아래와 같이 피가 네 가지 단계로 구분한다 : 혈소판과 혈장, 단핵 세포 (w하이트 링) 밀도 구배 용액 과립구 및 적혈구. </li><li> 새로운 50 ML 튜브에 플라스틱 파스퇴르 피펫 및 전송에 기본 백혈병 세포를 나타내는 흰색 반지를 수집합니다. <ol><li> PBS로 튜브를 실온에서 10 분 동안 300g에 총과 원심 분리기에 50 ㎖까지 (칼슘과 마그네슘을 포함)를 입력합니다. </li><li> 5 PBS 후 RT에서 10 분 동안 300g로 원심 분리하고 총 50 ㎖의 PBS까지 추가 ml로 펠렛을 재현 탁. </li><li> 완전한 RPMI 배지로 펠렛을 재현 탁 (RPMI 1640, 10 % 소 태아 혈청 (FBS), 2 mM의 글루타민, 100 U 보충 / ㎖ 페니실린 및 100 ㎎ / ㎖ 스트렙토 마이신) 세포 생존 카운터를 이용하여 카운트. </li></ol></li><li> 대기음는 상위 단계 과립구와 적혈구 위상을 떠나 25 ml의 최대 PBS를 추가는 총 50ml로 0.9 % NaClup에 3 % 덱스 트란을 추가합니다. 튜브 10 번을 혼합하고 혼합하지 않고 실온에서 30 분 동안 보관하십시오. </li><li> 상단 RBC 부족한 호중구 층를 수집깨끗한 멸균 50 ㎖ 튜브로 배치하고 RT에서 10 분 동안 300g로 원심 분리 튜브 거라고. PBS 후 총 50 ㎖까지 적혈구 용해 완충액을 추가 5 ㎖ 각 펠렛을 재현 탁. </li><li> RT 후 RT에서 10 분 동안 500g으로 원심 분리기에서 15 분 동안 어두운 데에서 튜브를 유지한다. PBS로 알약을 (총 50 ㎖까지 PBS를 추가) 다음 실온에서 10 분간 500 g에서 원심 분리 세척 할 것. </li><li> 세포 생존 카운터를 이용하여 카운트 완전한 RPMI 배지 펠렛을 재현 탁. 유세포 사용하여 분리의 순도를 결정하기 위해 5 ㎖ 플라스틱 튜브에 50 ㎕의 PBS-FBS (4 %)에서 각 세포 유형의 3 × 105을 유지한다. </li></ol></li><li> 분리 된 세포 집단의 형태 학적 출연 분석 <ol><li> 이를 위해, 각각 3 × 104 호중구 150 μL 완전 RPMI 배지에서 3 × 104 단핵 세포를 재현 탁. 즉 t을 보장 cytospin 원심 분리기에서 유리 슬라이드 필터 카드 및 샘플 챔버를 조립그는 원심 분리기는 균형이다. </li><li> 원심 분리기에서 슬라이드, 필터 카드 및 샘플 실을 제거 실온에서 10 분 동안 750 XG에서 샘플 챔버 및 사이토 원심 분리기로 각 셀 유형을 놓습니다. 슬라이드의 세포를 손상시키지 않도록 신중하게 분해한다. 필터 카드 및 샘플 챔버를 폐기하십시오. </li><li> 김사 염색 키트를 사용하여 세포를 염색하고 1 시간 건조 할 슬라이드를 유지합니다. 100X 배율 현미경으로 세포를 검사합니다. </li></ol></li><li> FACS에 의해 고립 된 세포의 순도 테스트 <ol><li> APC에 접합 된 항 - 인간 CD19 항체 (5 μL / 10 6 세포)와 격리 된 차 백혈병 세포를 레이블. 표 1 (5 μL / 10 6 세포)에 나열된 항 인간 항체의 혼합물로 정제 된 호중구 레이블. 4 O ℃에서 30 분 동안 어둠 속에서 세포를 부화 </li><li> 500 ㎕의 PBS-FBS (4 %) RT에서 5 분 동안 300g의 튜브를 원심 분리기로 세포를 씻으십시오. </li><li>200 ㎕의 PBS-FBS (4 %)의 각 펠렛을 재현 탁. </li><li> , SSC-A (488 /이 10bp), FITC (BP 530/30)를 PerCP-Cy5.5 (40분의 695 BP FSC-A (488 nm의) : 488 nm의 레이저 : 다음 광학 구성을 사용하여 사이토 흐름 분석 ), PE (BP를 575/26); 633 nm의 레이저 : APC (BP를 660/20), APC-Cy7 (BP를 780/60). 색 보정을 확인합니다. </li></ol></li><li> 자가 호중구와 차 백혈병 세포의 공 배양 <ol><li> 시드 차 백혈병 세포 단독으로 또는 완전 RPMI 배지에서 비 1:10자가 호중구로 2 × 105 세포 / ml. 이렇게 희석하여 세포 현탁액을 세포 수를 조절하고 2 × 106 세포 / ml로 호중구 주 백혈병 세포를 2 × 105 세포 / ㎖를 추가한다. 조심스럽게 섞는다. </li><li> 세포에의 Bruton의 티로신 키나제 (BTK) 억제제 (ibrutinib)의 25 μM을 추가합니다. 5 % CO 2와 37 ° C에서 24 시간 동안 품어. </li></ol></li><li> 세포 수확 및 FACS 분석 <ol><li> 콜요법 FACS 분석 및 원심 분리기 RT에서 5 분 동안 300g의 튜브 5 ML의 플라스틱 튜브에 세포. 다음 실온에서 5 분간 300 g에서 원심 분리기 1 ml의 PBS-FBS (4 %)와 펠렛을 씻으십시오. </li><li> 상업 키트를 사용하여 실온에서 10 분 동안 어둠 속에서 세포를 배양 재현 탁의 넥신 V의 펠렛 및 PI. (FSC-A (488 ㎚), SSC-A (488 /이 10bp), FITC는 (BP 530/30), PI 610 : 488 nm의 레이저 :도 2A의 게이트 전략을 사용하여 유세포과 같은 광학 구성에 분석 / 20 BP). 색 보정을 확인합니다. </li></ol></li></ol> 과립 통로 및 3D 모델에서 RL 림프종 B 세포와의 공동 배양을 따라 HL60 세포의 분화 (2) <ol><li> 호중구와 같은 세포로 인간 전 골수성 (HL60) 세포의 분화 <ol><li> / ㎖가 그들의 분화를 유도하기 위해 1 μm의 레티노 산 (RA) 및 1.25 %의 DMSO를 추가 3 × 10 5 세포에 HL60 세포 수를 조정합니다. 각 3 × 10 5 종자 </s최대&gt;도 48 웰 플레이트 당 HL60 세포는 5 % CO 2, 37 ℃에서 48 시간 동안 배양한다. </li><li> 그 후, 실온에서 5 분 300g에 세포와 원심 분리기를 수집합니다. 세포 생존 카운터를 이용하여 카운트 완전한 RPMI 배지로 세포를 재현 탁. </li><li> 이어서 용액 3 × 105 세포 /으로 HL60 세포의 수를 조정하고 1 μM 레티노 산 (RA) 및 1.25 % DMSO를 첨가하여 다시 분화를 유도하는 완전 RPMI 배지에서 세포 현탁액을 희석. 당 잘 48 웰 플레이트에 각 3 × 10 5 HL60 세포를 종자와 5 % CO 2와 37 ° C에서 또 다른 48 시간 동안 배양한다. </li></ol></li><li> HL60 분화 분석 (HL60의 차이) <ol><li> 세포를 수집하고 RT에서 5 분 동안 300g로 원심 분리 튜브. 세포 생존 카운터를 이용하여 카운트 완전한 RPMI 배지 펠렛을 재현 탁. </li><li> 유동 세포 계측법에 의한 세포 표면 마커의 발현에서의 변화를 분석. 각 3 × 10 (5)를 배치 </suP&gt; 미분화 (세포 배양에서) HL60 세포와 FACS 분석 및 원심 분리기 RT에서 5 분 동안 300g의 튜브 5 ML의 플라스틱 튜브에 3 × 10 5 HL60은 diff 세포. </li><li> 50 ㎕의 PBS-FBS (4 %)의 펠렛을 재현 탁. AF700 또는 APC에 접합 된 항 - 인간 CD38 (5 μL / 10 6 세포)에 접합 된 항 - 인간 CD11b를 가진 세포를 레이블. 4 O ℃에서 30 분 동안 어둠 속에서 세포를 부화 </li><li> 다음 실온에서 5 분 300g에 원심 분리기 500 μl의 PBS-FBS (4 %)로 세척. 200 ㎕의 PBS-FBS (4 %)의 펠렛을 재현 탁. </li><li> 488 nm의 레이저 :도 3A의 게이팅 전략과 같은 광학 구성을 이용하여 유동 세포 계측기에 분석 FSC-A (488 나노 미터), SSC-A (488 /이 10bp); 633 nm의 레이저 : APC (660/20 BP), 알렉사 플 루어 700 (BP를 730/45). </li><li> HL60은 diff의 형태 학적 변화를 테스트하려면, 현미경 검사 용 유리 슬라이드에 세포를 고정. <ol><li> 이렇게하려면 각 3 × 10을 재현 탁 <su(세포 배양)에서 P&gt; 4 미분화 HL60 세포 150 μl를 완전 RPMI 배지에서 3 × 10 4 HL60은 diff. 원심 균형 것을 보장 cytospin 원심 분리기에서 유리 슬라이드 필터 카드 및 샘플 챔버를 조립한다. </li><li> 실온에서 10 분 동안 1,500 rpm으로 샘플 챔버 및 사이토 원심 분리기로 각 셀 유형을 놓습니다. 원심 분리기에서 슬라이드, 필터 카드 및 샘플 실을 제거합니다. 슬라이드의 세포를 손상시키지 않도록 신중하게 분해한다. 필터 카드 및 샘플 챔버를 폐기하십시오. </li><li> 김사 염색 키트를 사용하여 세포를 염색하고 1 시간 건조 할 슬라이드를 유지합니다. 100X 배율 현미경으로 세포를 검사합니다. </li></ol></li></ol></li><li> 3 차원 (3D) 문화 주 :이 실험에 사용 된 물질은 저온이며 실험 얼음 일어나고 있는지 확인. <ol><li> 단독으로 또는 HL60은 diff와 혼합하거나, 각 5 × 10 4 RL 세포를 재현 탁 </em&gt; 비 RL에서 : HL60의 DIFF 1:10 약 2 내지 3 mm의 개구부를 넓게하기 위해 멸균 가위로 절단 한 ml의 피펫 팁을 사용하여 300 ㎕의 기저막 매트릭스. 이 단계에서 거품을 피하십시오. </li><li> 24 웰 플레이트에 잘 / 세포 현탁액 각각 300 μl를 시드. 이 단계에서 거품을 피하십시오. 그 다음 각 웰에 대해 RPMI 배지를 완료하고, 5 % CO 2, 37 ℃에서 7 일간 배양한다 ㎖로 1을 추가 5 % CO 2, 37 ℃에서 30 분 동안 플레이트를 배양한다. 매 2 일 매체를 변경합니다. 하루 5에서 10 나노 빈 크리스틴을 추가합니다. </li></ol></li><li> FACS 분석 <ol><li> 문화 7 일 후, 매체를 대기음 두 번 1 ml의 얼음처럼 차가운 PBS로 각 웰을 씻는다. 3 ㎖ / 얼음처럼 차가운 PBS-EDTA (5 mM)을 잘을 추가합니다. 200 μL 피펫 팁의 바닥을 이용하여 긁어 웰의 바닥에서 겔을 분리. 30 분 동안 얼음에 부드럽게 판을 흔들어. </li><li> 멸균 15 ML 튜브에 세포 현탁액을 전송하고 IC에 부드럽게 튜브를 흔들또 다른 30 ​​분 동안 전자. 균일 한 세포 현탁액의 모양을 확인합니다. (이 경우가 아니라면, 다음 긴 시간 동안 세포를 흔들 이상의 PBS-EDTA 추가). </li><li> RT에서 10 분 동안 300 XG에서 튜브를 원심 분리기. PBS로 펠릿 후 RT에서 10 분 동안 300 × g으로 원심 분리로 세척 하였다. </li><li> PBS-FBS (4 %)으로 재현 탁 및 PE Cy7하고 APC에 접합 된 항 - 인간 CD38 항체 (5 μL / 10 106 세포)를 접합 항 - 인간 CD19 항체 레이블. 4 오 ℃에서 30 분 동안 어둠 속에서 품어 </li><li> 실온에서 5 분 동안 300g에 500 ㎕의 PBS-FBS (4 %) 원심 분리기 튜브로 씻으십시오. 상업용 키트를 사용하여 아 넥신 V 및 PI로 세포를 재현 탁. </li><li> 도 4a의 게이팅 전략을 사용 사이토 흐름을 분석하고 다음 광학 구성 : 488 nm의 레이저 : FSC-A (488 ㎚), SSC-A (488 /이 10bp), FITC (530/30 BP), PI (610 / 20 BP), PE-Cy7 (780/60 BP); 633 nm의 레이저 : APC (BP를 660/20). 색 보정을 확인합니다. </li></ol></li&gt; </ol>

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The authors have nothing to disclose.

밀도 구배 원심 분리 방법을 이용하여 고순도의 인간 혈액 호중구의 분리를 위해 동일한 단계 단핵 세포 내에서, 여기에 효과적인 간단한 빠르고 저렴 프로토콜 설명도 분리 회수된다. 분리 된 세포 집단은 ≥90 % 순수 있습니다. 여러 가지 방법 인간의 혈액에서 호중구의 분리에 사용할 수 있습니다. 이러한 불연속 그라디언트 (11, 12)를 사용하거나 호중구는 면역 자?...

선천성 면역 세포는 종양 미세 환경 내에서 세포의 중요한 비율을 구성하는 환자, 암 (1)의 동물 모델에서 종양의 암과 연관되어왔다. 최근에,보다 널리 만성 면역 반응은 화학 요법에 대한 종양이 진행, 전이 저항 증진에 중요한 역할을하는 것으로 인식되고있다. 식세포 직접 화학 요법에 3,4- 종양 세포 반응을 조절하는 것으로 나타났다 중요한 선천성 면역 세포이다. 그러나 항암 치료에 대한 종양 반응을 조절하는 호중구의 선천 면역 키 선수의 역할은 알려져 있지 않다. 이러한 프로토콜의 목적은 CLL 환자의 혈액 샘플로부터의 호중구를 분리하는 빠르고 신뢰할만한 방법을 사용하여 안티 림프종 에이전트 림프종 세포의 민감도를 조절하는 역할을 연구하기 위하여 과립 통로 따라 HL60 세포를 구별한다. 6 침입에 대한 방어의 첫 번째 줄 혈액 (5) 행동의 선천성 면역 시스템의 가장 풍부한 세포의 구성 요소입니다. 호중구 여러 병리 적 상태 (7)에 가변 이펙터 기능 이외에 유효 선천성 면역 반응을 상승에 필수적인 역할을한다. 따라서, 이러한 농도 구배 분리법과 같은 다른 혈액 세포에서 호중구를 분리하는 빠르고 신뢰할만한 방법은 시험 관내 연구에 필요하다. 호중구 격리를 위해이 방법을 사용하면 생체 내 및 생체에서 호중구 매개 면역 기능에 대한 추가 연구를 촉진 할 것이다. 호중구 순수한 집단을 수득하는 능력은 면역 학적 질환 환자의 팔 조사 중요한 첫 단계이다. 밀도 구배 분리 방법은 세포의 높은 수율을 수득하는 이상적인 방법이다. 운전 방식D는 쉬지 않고 35 분 동안 300g으로 원심 분리 한 다음 희석 된 인간 혈액을 함유하는 튜브의 바닥에서 밀도 구배 용액의 첨가를 포함한다. 단핵 세포의 링 인터페이스에 표시하고, 호중구 전 이하 상주. 이 방법은 훨씬 더 9 비싼 호중구 분리 키트로 사용 가능한 다른 방법에 대한 상당한 이점이있다. 또한, 인간의 호중구에 대한 특정 표면 마커에 관한 항체를 사용하여 상업용 키트에 의해 인간 혈액으로부터 분리 호중구 세포 활성화 또는 분화의 위험을 증가시킨다. 밀도 구배 분리 방법은 단시간 호중구의 분리를 허용한다. 동일 단계 내에서 단핵 세포는 분리 회수된다. 순수한 세포의 높은 수율 무결성 기능을 달성하기 위해 수득되는 기본 기술이다. 종양 microenv을 모방하기 위해ironment, 3 차원 실험을 수행 하였다. 체외에서 호중구의 짧은 반감기를 고려할 때, 신선한 인간의 호중구와 3D 실험은 결정적인 없습니다. 이런 이유로, 전 골수성 (HL60) 세포 분화 유도제 디메틸 설폭 사이드 (DMSO) 및 레티노 산 (RA)를 사용하여 호중구와 같은 세포로 분화 유도된다. 차별화 된 HL60 세포 (HL60은 diff)를 사용하여 인해 다른 기증자로부터 격리에 호중구의 다른 응답을 갖는 것을 방지 할 수 있습니다. 체외 차원에서 문화 모델은 체외 2D 모델 사이의 생체 내 모델에서 중간 단계를 나타냅니다. 2D 문화에서, 세포는이 합성 표면에 변성되어 퇴적 단백질에 자연스러운 세포의 첨부 파일을 형성하는 플라스틱 표면에 확산. 반대로, 세포가 세포 외 기질의 합성 보낸 3D 배양 형태 천연 세포 - 세포 부착에 세포는 이들이 결합되어있는 천연 물질이다.이러한 이유로, 특히 암 세포 및 다른 세포 유형 간의 3D 공동 배양 모델은 종양 성장, 혈관 신생 및 전이에 대한 기여를 나타내는 매우 유용한왔다. 결과적으로, 3 차원 배양은 세포 배양 물이 생체 내 (10)에 존재하는 생리적 조건을 모방 할.

<table><tbody><tr><td>RPMI 1640&amp;nbsp;</td><td>Gibco Invitrogen</td><td>21875-034</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10270-106</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS contains calcium and magnesium)</td><td>Gibco Invitrogen</td><td>14040-091</td><td></td></tr><tr><td>N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td><td>Life technologies</td><td>25030-024</td><td></td></tr><tr><td>Penicillin streptomycin (Pen Strep)</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>Bruton&#039;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&amp;nbsp;</td><td>CliniSciences</td><td>A3001</td><td></td></tr><tr><td>Vincristine&amp;nbsp;</td><td>EG labo</td><td></td><td></td></tr><tr><td>BD Matrigel basement membrane matrix&amp;nbsp;</td><td>BD Biosciences</td><td>354234</td><td>Put at 4 &lt;sup&gt;o&lt;/sup&gt;C overnight (O/N) before the day of the experiment</td></tr><tr><td>Red cell lysis buffer&amp;nbsp;</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>Ficoll (Pancoll)</td><td>PAN Biotech</td><td>P04-60500</td><td></td></tr><tr><td>Dextran&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma-Aldrich</td><td>R2625</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Euromedex</td><td>S3014</td><td></td></tr><tr><td>Annexing V-FLOUS staining kit&amp;nbsp;</td><td>Roche</td><td>11&amp;nbsp;988&amp;nbsp;549&amp;nbsp;001</td><td></td></tr><tr><td>Kit RAL 555 Modified Giemsa staining kit</td><td>Cosmos Biomedical</td><td>CB361550-0000</td><td></td></tr><tr><td>LSRII flow cytometry</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Leica DMR-XA microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Cellometer Auto T4 Cell Viability Counter</td><td>Nexcelom&amp;nbsp; Bioscience</td><td></td><td></td></tr></tbody></table>

neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

흰색 환 단핵 세포이고, 혈소판 혈장 :. 여기에 설명 된 농도 구배 분리법은 CLL 환자의 혈액에서 분리 된 일차 백혈병 세포 및 자극되지 호중구도 1a는 위에서 아래로 (밀도 구배 원심 분리 후의 다른 혈액 층을 나타낸다 제공 밀도 구배 용액, 과립구 및 적혈구).도 1b 및 1c는 각각 호중구 (멀티 로브 핵 세포)와 단핵 세포의 형태 학적 외관의 차이를 ?...

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

neutrophil-isolation-analysis-to-determine-their-role-lymphoma-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.2K 조회수.  Cancer Research Center in Lyon. Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents. 

비디오: Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

호 중구 Extracellular 함정 밀도가 낮은 호 중구에 의해 생성 된 종양 세포 성장 및 첨부 파일 중재 하십시오 복 막 게 액체에서 얻은

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

연구

암 연구학

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

만성 림프모구 백혈병에서 소설 NFAT2 대상 유전자를 식별 하는 Chromatin Immunoprecipitation 분석 결과

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

기본 백혈병 세포의 신진 대사 프로필의 평가

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC 기반 분석은 인간의 만성 림프 구성 백혈병 세포에 세포 외 뉴클레오티드 / 뉴 클레오 시드 신진 대사를 모니터링하기

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

화학

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

핵/세포질 단백질 인신 매매를 모니터링하기 위한 1차 만성 림프구성 백혈병 세포의 세포 분열

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

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 ...

면역학 및 감염병학

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

항 종양 특성을 가진 분리 및 호중구의 특성

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

단백질 기능에 2DE - 젤 지점에서 만성 림프 구성 백혈병에 HS1에서 배운 레슨

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

의학

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

최적화 된 프로토콜은 림프구에 해당 작용과 미토콘드리아 호흡을 분석하는

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood.
The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x&#160;g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at &#8805;93% of viable cells for both types.
This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.

Here, we provide a reliable approach for isolating low- and normal-density neutrophils from whole blood using magnetic isolation (negative selection) and discontinuous density gradient medium. It ensures untouched isolation of high-purity cells (&#8805;93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.