ZG는 이스라엘 과학 재단 (부여 번호 11분의 41)의 I-CORE 프로그램에서 보조금 지원, Abisch-Frenkel 재단, Rosetrees 신뢰, 이스라엘 암 연구 재단 (ICRF - 연구 경력 개발 상) 및 관심사 재단. ZGF가 이스라엘 암 연구 재단 (ICRF - 연구 경력 개발 상)에서 보조금 지원, 건강의 이스라엘 정부와 이스라엘 폐 협회의 수석 과학자.

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+chemokines+secreted+by+tumor+cells+mount+a+lung+antimetastatic+response+during+renal+cell+carcinoma+progression.">Neutrophil chemokines secreted by tumor cells mount a lung antimetastatic response during renal cell carcinoma progression.</a> Oncogene. 32, 1752-1760 (2013).</li><li>Boxio, R., Bossenmeyer-Pourie, C., Steinckwich, N., Dournon, C., Nusse, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow+contains+large+numbers+of+functionally+competent+neutrophils.">Mouse bone marrow contains large numbers of functionally competent neutrophils.</a> J Leukoc Biol. 75, 604-611 (2004).</li><li>Sagiv, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+Diversity+and+Plasticity+in+Circulating+Neutrophil+Subpopulations+in+Cancer.">Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer.</a> Cell Reports. , (2015).</li></ol>

The authors have nothing to disclose.

호중구는 모든 백혈구 가장 풍부하며, 감염 및 염증의 경우 최초 대응한다. 이와 같이, 이들은 외부 큐에 매우 민감하고 쉽게 활성화된다. 또한, 호중구는 매우 짧은 반감기 및 빠른 회전율을 갖는다. 함께, 이러한 특성은 독특한 실험적인 전략이 필요되도록 호중구, 작업에 여러 가지 어려움을 올립니다. 예를 들어, 여러 호중구 정화 전략은 자신의 장점과 단점 각이있다. 호?...

호중구는 처음에 침입하는 미생물에 대한 첫 번째 줄에 방어 역할을 타고난 면역 세포 특징했다. 오늘은, 호중구가 더 많은 기능을 광범위한 것으로 알려져있다가, 외국 항원 1, 2에 적응 면역 반응을 장착 조혈 3, 4 혈관 신생을 조절하는 5 상처 치유에 관여된다. 또한, 호중구는 프로 및 항 종양 활동 -6,7-의 덕에 의해 종양의 성장 및 전이성 진행에 영향을 미칠 수있다. 호중구는 다형성 분할 핵을 특징으로한다 (따라서 다형 (PMN) 백혈구라고합니다) 적어도 세 과립의 별개의 서브 클래스뿐만 아니라 분비 소포 8 (그림 1A-C)를 포함한다. 호중구는 식균 높은 용량 및 미생물 제거를위한 임계 높은 NADPH 옥시 다제 활성을 갖는 한에 중요한 케모카인의 다양한 분비추가 호중구 염증 8,9의 사이트에 다른 면역 세포의 견인. 호중구는 수신자와 같은 수용체 (TLR들)을 포함한 표면 수용체의 대량 발현을 특징으로, C 형 렉틴 수용기 ​​(CLRS)는, 수용체 3 (하여 CD11b / CD18)과 다른 부착 분자 (예를 들어, L 셀렉틴을 보완 LFA-1, VLA-4 및 carcinoembryonic 항원 관련 세포 부착 분자 (3) (CEACAM3 / CD66b)), 케모카인 수용체 (예, CXCR1, CXCR2, CCR1, CCR2), 화학 유인 물질 수용체 (예, PAFR, LTB 4 R 및 C5aR) 사이토 카인 수용체 (예, G-CSFR, IL-1R, IL-4R, IL-12R, IL-18R, TNFR)은 수용체 (예 FPR1-3) 펩타이드를 포르 밀, 및의 Fc 수용체 (예를 들어, CD16 (FcγRIII 인간의 호중구는 CD11b를, CD15, CD16와 CD66b 백혈구 마커를 사용하여 식별되는 반면), CD32 (FcγRII) 및 CD64 (FcγRI) 10. 마우스에서 호중구는 일반적으로는 CD11b + Ly6G +로 식별됩니다. 또한 일반적으로 조직에서 호중구의 검출을위한 과립 단백질 된 myeloperoxidase (MPO) 및 호중구 엘라 스타 제 (NE)에 대한 얼룩 허용됩니다. 그것은 호중구의 다양한 기능이 동일하거나 별개의 셀에 의해 세포의 서브 모집단에 의해 매개되는지 아직 불분명하다. 축적 된 데이터는 염증성 자극과 미세 환경의 영향 11,12 가소성의 높은 정도를 나타내는 이종의 인구 호중구의 존재를 시사한다. Fridlender 등. (13)는 크게 프로 종양 특성을 가진 항 종양 특성 및 N2로 N1 불리는 두 개의 주요 하위 집단으로 암의 호중구를 분할했다. 암뿐만 아니라 만성 염증, T 세포 반응을 억제 과립 14 골수 유래 세포 억제제 (G-MDSCs)로 구성된 부가적인 서브 집단이있다. G-MDSCs이는 CD11b + Ly6C 특징으로 미성숙 골수 세포로 간주됩니다쥐 15 낮은 Ly6G 안녕하세요 표현형, 인간의 16 CD15 + / CD16 낮은 표현형을하면서. G-MDSCs 정상 순환 호중구보다 사이토 카인과 케모카인의 동안 낮은 수준을 아르 기나과 마이 엘 로퍼 옥시 다제의 높은 수준을 표현한다. 그들은 이하 식세포 및 이동성을하지만 ROS 15,17,18 높은 수준의 생산. 본 논문에서는 항 종양 특성을 가진 호중구의 분리 및 특성에 대한 몇 가지 기본적인 방법을 설명합니다. 호중구는 인간의 순환에서 모든 백혈구의 가장 큰 인구를 구성하는 동안 (45~70%; 1,800 - 6,000 / μL) -; 300-500 / μL 15 %, 생쥐에서 정상 조건 하에서, 그들은 10 (오히려 드문 드문 ). 염증에 따라 때때로 만성 염증 (7)의 상태를 나타내는 암에 지속적으로 호중구 수가 증가한다. 호중구는 다 능성 일반적인 골수 전구체 (CMP) CE에서 개발골수성의 단계 (메가 바이트), promyelocytes (오후), myelocytes (MC), metamyelocytes (MM) 및 밴드 세포 (BC)를 통과하는 분화 과정을 통해 골수의 재편, 8. 그들은 순환 (8)에 공개되기 전에 7 일 - 성숙, 후 유사 분열 호중구는 4 골수 내에 남아있을 수 있습니다. 염증 상태에 따라 연장 될 수있다 12 시간 - 혈중 호중구 매​​출 6의 평균 반감기 일반적으로 빠르다. 비자극 호중구는 포르 볼 에스테르 포르 볼 (12)에 노출시킴으로써 항 - 종양 활성, 케모카인 IL-8 (CXCL2), CCL2, CCL5 및 CXCL5 6,19 또는 인위적으로 나이브 호중구를 노출시킴으로써 얻어 질 수있는 기능을 제한적 -myristate 13 아세테이트 (PMA) 6. 호중구의 낮은 번호와 함께 혈액 호중구의 짧은 반감기 (~ 3-5 10 5 ×) 순진한 (6) 1 ml의 혈액에서 달성 - 8 주 이전 마우스, 그것을 만든어려운 시험 관내에서 마우스 호중구 순환의 기능을 탐구한다. 이러한 어려움을 극복하기 위해, 다른 소스가 이용되고있다. 예를 들면, 호중구의 다수는 (티오 글리콜 레이트 브로 쓰 또는 자이 모산의 복강 내 주사 한 후, 예) 골수 20 무균 염증 유도 다음 복막로부터 획득 될 수있다. 이는 복강 호중구로부터 얻어지는 모든 항 종양 활성 (미공개 관찰)을 발휘하는 것은 아니다. . 40,000,000 혈액 호중구 용이 1 mL의 혈액으로부터 분리 할 수있다 - Granot 외 6은 BALB / c 마우스 (20)가되도록 종양 진행 (6) (도 2A)으로 악화 호중구를 개발 마우스 4T1 유방 암종 세포주 동소 접종 관찰 3-4주 후 종양 접종. 이러한 호중구 항암 활동을 획득하고 이에 따라 COI왔다순서 네드 종양 동반 된 호중구 (TEN)는 순진한 호중구 (6) (그림 2B)와 구별한다. 고밀도 호중구 (HDN,도 1a)가 높은 항 종양이지만, 암의 문맥에서 생성 저밀도 호중구 (LDN,도 1b) (21)는 그렇지 않다. 또한, 종양 - 함유 마우스의 골수 및 비장으로부터 고밀도 호중구는 항 종양 활성 (미공개 데이터)를 갖는다. 그것은 호중구의 양의 증가와 함께, 종양 진행과 비장 서서히 확대 (비장)을가되도록 유의해야한다. 그것은 10도 자연 (MMTV-PyMT 및 MMTV-Wnt1 유방 종양과 K-RAS 중심의 폐 종양) 모두를 포함하는 암의 다른 모델에서 생성 및 AT-3 (MMTV-PyMT)과 E0771의 유방암 (주입 주목해야한다 세포, LLC 루이스 폐 암종 세포 및 B16-F10 흑색 종 세포). 이 다치거나에서 호중구 동원하지만, 범위삼주 후 1 ml의 혈액에서 10 × 10 6 호중구 - 또는 모델 5에 도달, 4T1-접종 생쥐보다 훨씬 적습니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse 4T1 breast carcinoma cells</td>
			<td>ADCC</td>
			<td>CRL-2539</td>
			<td>Growth medium: DMEM + 10 % heat-inactivated FBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLASTIC WARES AND EQUIPMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Tissue Culture Plate&#160;</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture Plate&#160;</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25 cm2 Tissue Culture Flask</td>
			<td>Nunc</td>
			<td>156340</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>90 mm Bacterial Grade Culture Dish&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>20090-01-017</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>15 ml Sterile Conical Centrifuge Tube&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>835015-40-111</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>50 ml Sterile Conical Centrifuge Tube&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>835050-21-111</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Falcon 12x75 mm Round-Bottom Polystyrene Tube&#160;</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Millicell 24 Migration Plate with a pore size of 5&#956;m&#160;</td>
			<td>Merck Millipore</td>
			<td>PSMT010R1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>White 96-Flat-Bottom Well Plate&#160;</td>
			<td>Costar</td>
			<td>3917</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Cell Strainer (40 mm)&#160;</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>20G 1.5&#34; Needle</td>
			<td>BD Microlance 3&#160;</td>
			<td>301300</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>23G 1&#34; Needle&#160;</td>
			<td>BD Microlance 4</td>
			<td>300800</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25Gx5/8&#34; Needle&#160;</td>
			<td>BD Microlance 5</td>
			<td>300600</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>0.3 ml Syringe with a 30Gx8mm Needle</td>
			<td>BD Micro-Fine Plus Demi</td>
			<td>320829</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>9 mm Clips&#160;</td>
			<td>BD, AutoClip&#160;</td>
			<td>427631</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>EasySep Magnet&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS LS Separation Column&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-201</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>MidiMACS Separator Magnet</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Glass Slide&#160;</td>
			<td>Menzel-Gl&#228;ser Superfrost&#160; Plus Thermo</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker&#160;</td>
			<td>Sky line, ELMI</td>
			<td>S-3.02.10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Reader&#160;</td>
			<td>TECAN</td>
			<td>InfiniteF200Pro</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>POWDER</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), fraction V</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU)&#160;</td>
			<td>BD Pharmingen</td>
			<td>550891</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>CFSE (5-(and 6-)-Carboxyfluorescein diacetate, succinimidyl ester)</td>
			<td>Molecular Probes</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Sigma</td>
			<td>31392</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa&#160;</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>Sigma</td>
			<td>S8032</td>
			<td>Highly toxic, handle with care</td>
		</tr>
		<tr>
			<td>Thioglycollate powder&#160;</td>
			<td>Difco</td>
			<td>225650</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymosan A</td>
			<td>Sigma</td>
			<td>Z4250</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEDIA AND SUPPLEMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)</td>
			<td>Sigma</td>
			<td>D5796</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Opti-MEM&#174; I reduced serum medium&#160;</td>
			<td>Life Technologies</td>
			<td>31985062</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI)-1640 medium</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS), heat-inactivated</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-020-1A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-042-1B</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin x1000 solution</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-031-5</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) without Mg2+ and Ca2+&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-023-1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>PBSx10 without Ca2+ and Mg2+&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-023-5A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>HPLC grade water&#160;</td>
			<td>J.T. Baker</td>
			<td>4218-03</td>
			<td>Autoclave</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SOLUTIONS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACK &#8211; Ammonium-Chloride-Potassium</td>
			<td>Life Technologies&#160;</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU) solution (10 mg/ml) in PBS</td>
			<td></td>
			<td></td>
			<td>Dissolve 10 mg of BrdU in 1 ml PBS and sterile filter.</td>
		</tr>
		<tr>
			<td>CFSE, 5 mM in DMSO</td>
			<td></td>
			<td></td>
			<td>Dissolve 2.8 mg of CFSE in 1 ml DMSO. Divide into 10 ml aliquots in sterile 200 ml tubes and store in the dark at -20oC.</td>
		</tr>
		<tr>
			<td>Eosin Y solution</td>
			<td>Sigma</td>
			<td>HT110-2-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt solution</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-016-1A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Heparin, 20 mg/ml in PBS</td>
			<td></td>
			<td></td>
			<td>Dissolve 100 mg Heparin in 5 ml sterile PBS, and sterile filter through a 0.2 mm filter.&#160;</td>
		</tr>
		<tr>
			<td>Histopaque-1119&#160;</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>Sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>Histopaque-1077&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td>Sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>&#160;Luciferase cell culture lysis buffer x5</td>
			<td>Promega</td>
			<td>E153A</td>
			<td>Dilute 1:5 in sterile water just before use.</td>
		</tr>
		<tr>
			<td>Luciferase assay solution</td>
			<td>Promega</td>
			<td>E1501</td>
			<td>Contains luciferase assay substrate powder (E151A) and luciferase assay buffer (E152A)</td>
		</tr>
		<tr>
			<td>Mayer&#39;s Hematoxylin solution&#160;</td>
			<td>Sigma</td>
			<td>MHS-32</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS+0.5% BSA</td>
			<td></td>
			<td></td>
			<td>Dissolve 2.5g BSA in 500 ml PBS, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>PBS+1% BSA</td>
			<td></td>
			<td></td>
			<td>Dissolve 1g BSA in 100 ml PBS, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>5x PBS with 2.5% BSA</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 12.5g BSA in a mixture of 250 ml sterile HPLC-grade water&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; and 250 ml PBSx10, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>PBS containing 0.5% BSA and 2 mM EDTA</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 250 mg BSA in 50 ml sterile PBS&#160; and add 200 ml of 0.5M EDTA pH 8.0, sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>FACS buffer (PBS containing 0.5% BSA, 2 mM EDTA and 0.02% NaN3)</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 250 mg BSA in 50 ml sterile PBS&#160; and add 200 ml of 0.5M EDTA pH 8.0 and 500 ml of 2% NaN3, sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>Saline (0.9% NaCl)</td>
			<td></td>
			<td></td>
			<td>Dissolve 9 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>0.2% NaCl solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 2 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>1.6% NaCl solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 16 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>2 % Sodium azide</td>
			<td></td>
			<td></td>
			<td>Dissolve 1g sodium azide in 50 ml sterile ddw, keep at 4oC. Highly toxic.</td>
		</tr>
		<tr>
			<td>3% Thioglycollate solution</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 3 g of thioglycollate powder in 100 ml ddw.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Boil until solution becomes yellow and autoclave.</td>
		</tr>
		<tr>
			<td>Trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>Dilute 1:10 in PBS to get a 0.04% solution.</td>
		</tr>
		<tr>
			<td>Trypsin solution B&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-046-1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>1 mg/ml Zymosan A</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Resuspend 1 mg Zymosan A&#160; in 1 ml sterile PBS in an Eppendorf tube.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Vortex vigorously and incubate the tube at 37 oC for 30 min. Do not autoclave.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Prepare the solution freshly before use.&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KITS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep PE selection kit</td>
			<td>STEMCELL Technologies</td>
			<td>18557</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep PE selection cocktail&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18151</td>
			<td></td>
		</tr>
		<tr>
			<td>the EasySep magnetic nanoparticles&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18150</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ly6G mouse MicroBead Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-332</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human Neutrophil Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19257</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC BrdU flow kit</td>
			<td>BD Pharmingen&#160;</td>
			<td>559619</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Neutrophil isolation kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis Assay Kit&#160;</td>
			<td>Cayman Chemical Company&#160;</td>
			<td>500290</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocking antibody&#160;</td>
			<td>Biolegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-Ly6G antibody&#160;</td>
			<td>BD Pharmingen&#160;</td>
			<td>551459</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Ly6G antibody&#160;</td>
			<td>Biolegend</td>
			<td>127608</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse Ly6G</td>
			<td>BD Pharmingen&#160;</td>
			<td>551460</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 rat anti-mouse Ly6G&#160;</td>
			<td>TONBO Biosciences</td>
			<td>65-1276</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>violetFluor 450-conjugated rat anti-mouse Ly6G&#160;</td>
			<td>TONBO Biosciences</td>
			<td>75-1276</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse CD11b</td>
			<td>BD Pharmingen&#160;</td>
			<td>553310</td>
			<td>Clone M1/70</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse Ly-6G and Ly-6C (GR-1)</td>
			<td>BD Pharmingen&#160;</td>
			<td>553127</td>
			<td>Clone RB6-8C5</td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse CD45</td>
			<td>BD Pharmingen&#160;</td>
			<td>553081</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse F4/80&#160;</td>
			<td>Abcam</td>
			<td>ab60343</td>
			<td>Clone BM8</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD66b&#160;</td>
			<td>Biolegend</td>
			<td>305103</td>
			<td>Clone G10F5</td>
		</tr>
		<tr>
			<td>Purified rat isotype control antibody (IgG2a, k)&#160;</td>
			<td>BD Pharmingen&#160;</td>
			<td>553927</td>
			<td>Clone R35-95</td>
		</tr>
		<tr>
			<td>LEAF purified Armenian hamster anti-mouse CD3e antibody</td>
			<td>BioLegend</td>
			<td>100314</td>
			<td>Clone 145-2C11</td>
		</tr>
	</tbody>
</table>

isolation and characterization of neutrophils with anti-tumor properties

호중구 순환 모든 인간 백혈구의 가장 풍부한는 침입 미생물에 대한 숙주 방어에 중요한 역할을한다. 또한, 호중구는 종양 세포의 면역 감시에서 중심적 역할을한다. 이들은 어느 과산화수소 또는 항체 - 의존성 세포 매개 세포 독성 (ADCC)을 통해 관련된 세포 의존성 접촉 메커니즘을 통해 종양 세포의 사멸을 종양 세포를 인식하고 유도 할 수있는 능력을 가지고있다. 항암 활성을 갖는 호중구 암 환자의 종양 - 함유 마우스의 말초 혈액으로부터 분리 할 수​​있다. 이러한 호중구는 현저한 종양 세포 상해 활성을 보여 정상인 또는 나이브 마우스의 호중구 구별하는 종양 혼입 호중구 (TEN)를 지칭한다. 다른 백혈구에 비해 호중구 밀도 구배 실시 때 실현&gt; 98 % 순수 호중구 집단을 얻을 수있다 다른 부력을 나타낸다. 그러나, 또한통상 고밀도 호중구 인구 (HDN)에, 암 환자에서 종양 - 함유 마우스에서뿐만 아니라 만성 염증성 조건 하에서 구별 저밀도 호중구 인구 (LDN)는 순환 나타난다. LDN는 단핵구 분획 공동 정제 및 양성 또는 음성 선별 전략을 사용하여 단핵 세포로부터 분리 될 수있다. 절연 호중구 순도 유동 세포 계측법에 의해 결정되면, 이들은 시험 관내 및 생체 기능 분석에 사용될 수있다. 우리는 호중구의 항 종양 활성을 모니터링하는 능력 마이그레이션 및 반응성 산소 종을 생성 할뿐만 아니라 생체 탐식 능력을 모니터링하는 방법을 설명한다. 우리는 또한 생체 내 추적 호중구 레이블을 기술을 설명하고 생체 내에서 자신의 안티 전이성 용량을 확인할 수 있습니다. 이러한 모든 기술은 획득 및 항 종양 호중구를 특성화하는 방법을 이해하는데 필수적기능.

최근의 연구에서 우리는 호중구 6 항 전이 기능을 확인 하였다. 종양 베어링 마우스에서 호중구는 세포 독성 표현형을 획득하고 종양 세포 (6)를 죽일 수있는 능력을 가지고있다. 이는 현저한 항 종양 효과가없는 6 나이브 마우스의 호중구는 대조적이다. 프로토콜 절에 설명 된 여러 기술은 시험 관내 및 생체 내 6 항 종양 호중구 기능 연구를 위해 사용되어?...

Watch this Scientific Journal Video about Isolation and Characterization of Neutrophils with Anti-Tumor Properties at JoVE.com

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

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 ...

isolation-characterization-neutrophils-with-anti-tumor

Research

JoVE Journal

Immunology and Infection

24.6K 조회수.  Hebrew University Medical School. 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. 

비디오: Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

바이러스 변이 주파수의 RNA 바이러스 및 특성화의 피델리티 변종의 분리

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

연구

면역학 및 감염병학

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

마우스 소장에서 돌기 세포 및 Macrophages의 분리 및 특성화

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

연쇄상 구균 폐렴으로 비강 내 식민지 화 중 염증 반응의 특성화

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

분리, 정제 및 기능 연구 및 입양 전송 실험 쥐의 골수 호중구의 라벨링

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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. ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

쥐 소장 림프구의 분리 및 유세포 특성

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

를 사용하여 림프구 혈관 외로 유출 분석<em&gt; 체외</em&gt; 인간의 혈액 - 뇌 장벽의 모델

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

절연 및 특성화 Neutrophil 파생 된 미의 기능 연구

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

복 막 파생 돛대 세포 및 그들의 기능 특성화 캘리포니아2 +의 격리-이미징 및 Degranulation 분석 실험

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...