저자는이 절차의 최적화 동안 그들의 도움을 잭 슈, 케이시 존, 린 Cialdella - 캄을 인정하고 싶습니다.

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The authors have nothing to disclose.

이 원고는 과립구 및 단핵구 함수의 두 개의 지표를 결정하기위한 단계적인 프로토콜을 제공한다. 우리는이 분석의 성공에 중요한 몇 가지 단계를 확인했다. 그러한 단계는 세균의 첨가 직전에 발생 빙욕 배양한다. 모든 샘플을 철저히 냉각 식세포 및 OB 활성에 대한 온도의 영향을 최소화한다. 다른 중요한 단계는 각각의 샘플에 대한 박테리아의 추가이다. 8 : 1 세균 : 식세포 비율이 최적의 결과를 생성한다; 그러나 다른 연구자들은이 비율이 적절한 결과를 산출하도록 수정 될 필요가 있는지 찾을 수있다. 또 다른 중요한 단계는 백혈구를 적혈구를 용균하고 해결하기 위해 RBC의 용균과 WBC 고정 시약의 사용이다. 이 같은 시약의 사용은 적혈구의 완전한 용해를 보장하고 안전하게 밤새 4 ℃에서 처리 된 샘플을 저장 한 후 다음날 흐름 cytometer 통해 실행할 연구자 수있다. 이전 작업 (미 출판) 인도어RBC의 용균 및 WBC 고정 데이터 무결성을 손상시키지 않고 많은 12 시간 고정하여 시료의 유동 세포 계측법을 지연시키는 축열. 이 절차는 용이 연구원의 능력과 흥미를 충족하도록 변경 될 수 있지만, 적응이 이루어질 때 최적화가 필요한 것이 강조되어야한다. 보고 된 가장 일반적인 적응 중 일부는 타겟 박테리아, 라벨 / 프로브 및 배양 시간한다. 대장균은 7,8 S. 대신에 사용될 때 연구자 뛰어난 결과를 생산 구균 대상 병원체로 (모두 8에서 : 1 비율). 이러한 사카 cerevisiae의 같은 균류는 또한 E. 대용으로 적합 할 수있다 대장균. 또한, 연구진은 자주 식균 작용을위한 프로브와 같은 pH 민감성 분자 프로브, 대신 FITC의 7,12를 사용합니다. HE 일반적 OB 활성을 측정하기 위해 사용되는 것보다 마찬가지로 includin 다른 프로빙g의 dichlorofluorescin 디 아세테이트 (DCF-DA) 10 123 dihydrorhodamine (DHR). (13)보고 배양 시간은 20 분에서 120 분, McFarlin은 최대 응답 시간이 박테리아 목표에 따라 다를 수 있다는보고와 12,14,15에 따라 다릅니다. 7 그것은해야 또한 최적의 배양 시간은 분석의 식균 작용 및 OB 측면 사이에서 변할 수도있다. (7) 새로운 연구 환경에서이 기술을 설정할 때 모든 실험실 분석과 마찬가지로, 문제 해결이 필요할 수 있습니다. 저자는 박테리아의 최적화 조언 : 식세포의 비율이 분석을위한 성공의 열쇠입니다. 저자는 또한 비 질병 참가자로부터 과립구 세포 모집단의 80 % 미만이 양 FITC하는 경우의 샘플 데이터를 분석 폐기되어야 함을주의; 이러한 상황에서, 샘플을 다시 회수 할 수 있고, 가능하다면 재 - 분석. 받는 몇 가지 제한 사항이 있습니다과립구와 단핵구 식균 작용 및 OB 활동 분석은이 논문에서 설명했다. 그러한 제한은 관심있는 세포 집단을 식별 FSC 및 SSC의 전용이다. 세포 표면 마커의 사용은 연구자들이 긍정적 세포군의 동정을 행 하였다. (10) 그러나,이 기능의 추가가 사이토 기본 흐름 이상을 필요로 허용하고,이 원고의 범위를 벗어나는 따라서 인 것이다. 이 분석의 또 다른 한계는 관심 면역 세포에 의해 내재화되지 않은 프로브의 형광을 종결 트리 판 블루의 효능에 의존한다는 것이다. pH를 성 분자 프로브는 7,12 내재화가 문제가되는 상황에서 FITC에 대한 대안으로 사용될 수있다. 이러한 프로브는 세포 내 이입 소포에서와 같은 산성 환경에서만 형광체이기 때문에, 획득 된 형광 내면화 입자만을 나타낸다. 이 않기 때문에 또한, 몇몇 연구자들은 현재의 분석을 비판ES 기준선 면역 세포 활성에 대한 제어 얼음 물 대신에 37 ℃에서 배양 된 샘플의 병렬 세트를 포함하지. 여러 경우에, 우리는 과립구 및 단핵구와 OB 탐식 활성 분석을 수행 할 때 &quot;얼음 상태&quot;를 포함하고 크게 (미공개) 데이터의 품질이 향상되지 않는 것을 알았다. 요약하면,이 원고는 과립구 및 단핵구에 식균하고 OB 활성을 측정하는 방법을 설명한다. 이것은 쉽게 실험실 능력 및 연구 분야를 설명하기 위해 수정 될 수있는 간단하고, 간단한 분석이다. 운동 과학 - 비만 관련 연구에서 선천성 면역 기능이 측정은 조사가 체중 감소, 보충 교재 사용,식이 개입 및 기타 생활 습관의 변화 등 많은 잠재적 대책의 효과를 탐구 할 수 있습니다. 이 기술을 마스터하면, 연구진은 급성 모니터링 할 수 있습니다다이어트, 운동, 그리고 많은 다른 자극에 응답하여 면역 세포의 기능 및 만성 변경됩니다.

과립구 및 단핵구 식세포 / 산화 버스트 (OB) 활성 분석은 종종 장시간 집중 운동 다음 선천성 면역 기능에 대한 정보를 수집하는 데 사용되는 간단한 직선적 기술이다. 1-4 면역계의 반응을 연구 할 때 이들이 숙주 방어에서 중요한 역할을 감염 부위에 축적 한 최초의 면역 세포이기 때문에, 자극, 과립구 및 단핵구는 특히 중요하다. 5 호중구 과립구의 일종 손상된 근육시키다 최초 셀은 집중 운동 다음 티슈. 6 탐식 및 OB 활성 과립구 및 단핵구 기능 (7)의 가장 일반적인 방법이며 때문에 감염 과정 및 복구 프로세스 모두에서의 중추적 인 역할의 선천성 면역 세포 기능의 지표로서 바람직하다. 이 원고 utili에서 설명하는 분석ZES 기본적인 플로우 사이토 전혈 과립구 및 단핵구와 OB 탐식 활성의 정량적 측정을 제공한다. 이 분석은 시간 소모적 정제 과정없이 수행 될 수 있기 때문에 이러한 기술에서 전혈의 사용은 유리하다. 이러한 분석을 용이하게 연구 설계 및 실험실 기능의 다양성을 수용하기 위해 수정 될 수있다. 예를 들어, 리아 등. 그 호중구 OB 활동을 표시하는 유사한 기술을 이용 외상성 뇌 손상 후 증가되고 호중구 식세포 감소된다. (8) 또 다른 예에서, McFarlin는 외. 알로 사용하는이 기술은 우아한 변형 (APC 바와 )는 식세포 작용 및 OB 활동의 관점에서 과립구 분류 이미지 기반 유동 세포 계측법으로 CD66b 고성능 직렬 APC 공역 CD45 라벨링 π 공역. 7,9 우리의 연구 그룹은이 테의 다양한 적응을 사용하고있다거의 20 년 동안 과체중 비만, 체육 인구의 선천성 면역 기능의 지표로 chnique. 10, 11 텍스트 아래이 분석을 수행하기위한 단계별 지침과 독자를 제공하고 독자가 적응할 수있는 영역을 강조합니다 연구의 요구를 충족한다.

<table><tbody><tr><td>12 x 75 mm tubes, with cap</td><td>VWR</td><td>20170-579</td><td>You will need 2 tubes per sample (&quot;H&quot; and &quot;F&quot;) plus 3 tubes per batch (for assay controls; &quot;F&quot;, &quot;H&quot;, and &quot;Blood only&quot;).</td></tr><tr><td>PBS (10x), pH 7.4</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Bottle top 0.2 &amp;micro;m cellulose acetate filter (500 ml capacity)</td><td>Fisher Scientific</td><td>09-741-07&amp;nbsp;</td><td></td></tr><tr><td>Glucose, powder</td><td>Life Technologies</td><td>15023-021</td><td></td></tr><tr><td>Citric acid (anhydrous, cell culture tested, plant cell culture tested)</td><td>Sigma-Aldrich</td><td>C2404-100G</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma-Aldrich</td><td>S4641-25G</td><td></td></tr><tr><td>Dihydroethidium (Hydroethidine) (HE)</td><td>Life Technologies</td><td>D11347</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO) Anhydrous</td><td>Life Technologies</td><td>D12345</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)</td><td>Life Technologies</td><td>S2851</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, unlabeled</td><td>Life Technologies</td><td>S-2859</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>4.0 ml vacutainer containing 7.2 mg K&lt;sub&gt;2&lt;/sub&gt;EDTA, spray-dried</td><td>VWR</td><td>BD367861</td><td>You will need 1 K&lt;sub&gt;2&lt;/sub&gt;EDTA blood collection tube per sample.</td></tr><tr><td>4.0 ml vacutainer containing 68 USP units Lithium Heparin, spray-coated</td><td>VWR</td><td>BD367884</td><td>You will need 1 Lithium Heparin blood collection tube per sample.</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff CP</td><td>Beckman Coulter</td><td>6605705</td><td>&lt;em&gt;i.e.&lt;/em&gt;, hematology analyzer</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Rinse</td><td>Beckman Coulter</td><td>8547167</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Fix</td><td>Beckman Coulter</td><td>8547171</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff WBC Lyse</td><td>Beckman Coulter</td><td>8547170</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Hgb Lyse</td><td>Beckman Coulter</td><td>8547168</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Cal Calibrator</td><td>Beckman Coulter</td><td>7547175</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Control Plus</td><td>Beckman Coulter</td><td>7547198</td><td></td></tr><tr><td>AcT 5diff Diluent</td><td>Beckman Coulter</td><td>8547169</td><td></td></tr><tr><td>200 &amp;micro;l extended-length pipette tips</td><td>VWR</td><td>37001-526</td><td></td></tr><tr><td>Insulated ice pan</td><td>VWR</td><td>89198-980</td><td>For ice water bath.</td></tr><tr><td>Open metal tube rack</td><td>VWR</td><td>60916-702</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>TQ-Prep Workstation</td><td>Beckman Coulter</td><td>6605429</td><td>&lt;em&gt;i.e.&lt;/em&gt;, automated cell lyse preparation workstation</td></tr><tr><td>ImmunoPrep Reagent System</td><td>Beckman Coulter</td><td>7546999</td><td>&lt;em&gt;i.e.&lt;/em&gt;, RBC lyse/WBC fix reagent system</td></tr><tr><td>Cytomics FC500 MCL Flow Cytometry System with CXP Software</td><td>Beckman Coulter</td><td>626553</td><td></td></tr><tr><td>IsoFlow Sheath Fluid</td><td>Beckman Coulter</td><td>8547008</td><td></td></tr><tr><td>COULTER CLENZ</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>Flow-Check Fluorospheres&amp;nbsp;</td><td>Beckman Coulter</td><td>6605359</td><td>&lt;em&gt;i.e.&lt;/em&gt;, alignment and fluidics verification fluorospheres</td></tr><tr><td>FlowJo Software</td><td>FlowJo</td><td>FlowJo</td><td>&lt;em&gt;i.e.&lt;/em&gt;, flow cytometry analysis software</td></tr><tr><td>Excel Software</td><td>Microsoft</td><td>Microsoft Excel</td><td>&lt;em&gt;i.e.&lt;/em&gt;, electronic spreadsheet program</td></tr></tbody></table>

measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

장기간 집중적 인 운동은 자연 살해 세포 기능, 바이러스 성 감염에 대 식세포 사이토 카인 매개 응답 및 과립구와 단핵구의 식균 작용 및 OB 활동을 포함한 선천성 면역 기능에 심각한 영향을 보유하고 있습니다. 여러 연구는 근육 손상 및 신진 대사 요구에 의해 유도 된 염증을 반영하는 과립구와 단핵 탐식의 대폭 증가 후 운동을 나타냅니다. 반면, 과립구와 단핵구 OB 활동은 운동 후 감소 및 감염에 대한 감수성 증가의 한 면역 지표가 될 수있다. 예를 들어, 그림 1은 S.의 식균 작용을 보여줍니다 과립구에 의해 구균은 다음 날 아침에 정상에 복귀, 75 km의 자전거 한판 승부 (70 % VO 2MAX) 후 즉시 1.5 시간 증가된다. 단핵 세포의 식균 작용은 유사 운동이 수준에 의해 영향을 받는다. 2는 그림 과립구 OB 활동의 감소가 있음적어도 21 시간 75 km 후 사이클링 라. 단핵구 OB 활동은 75 km 사이클링 후 감소된다. S.의 과립구와 단핵구 식균 작용 구균이 아니라 과립구와 단핵구 OB 활동, 전신 염증 바이오 마커에 ​​상관 관계 (3)는 과립구 식균 작용 및 혈청 C 반응성 단백질 (CRP) 수치 (피어슨 제품 순간 상관 관계 사이의 완만 한 상관 관계를 보여줍니다;. R = 0.44; P &lt;0.01 ) 106 과체중 / 비만 여성입니다. 혈청 CRP는 S.의 단핵 식세포 상관된다 구균 (피어슨 제품 순간 상관 관계; R = 0.30; P &lt;0.01). 또한, 혈중 호중구 / 백혈구 (피어슨 적률 상관 각각 R = 0.62이고 r = 0.61; P &lt;0.001), 체질량 지수 (피어슨 적률 상관; R = 0.50, R = 0.40, 각각; P &lt;0.001), 혈청 인슐린 농도 (R = 0.30, R =0.23; P &lt;0.03), 혈액 1C 수준 (피어슨 제품 순간 상관 각각 R = 0.28이고 r = 0.21; P &lt;0.04)은 S.의 과립구와 단핵 탐식에 상관 관계 구균. 일반적으로 이러한 데이터는 휴식 과목에서 높은 과립구와 단핵 세포의 식균 작용은 전신 염증을 나타내는 것을 나타냅니다. <img alt="그림 1" src="/files/ftp_upload/54264/54264fig1.jpg" /> 그림 1 :. 과립구 탐식은 강렬한 지구력 운동에 의해 영향을받는 과립구의 식균 작용은 VO 2MAX의 70 %에서 75 km의 자전거 시합 후 즉시 1.5 시간 증가된다. 21 시간 후 - 운동으로, 과립구 식세포 작용이 약간 운동 전 수준 아래에 돌아왔다. * 운동 전 다른 표시 (쌍 t-test를 사후 분석 반복 측정 ANOVA, 적절한 경우, N = 19, P &lt;0.05). 값은 나에게있다± 표준 오차. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54264/54264fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54264/54264fig2.jpg" /> 그림 2 :. 과립구 산화 버스트 활동이 강렬한 지구력 운동 과립구의 산화 버스트 활동에 의해 영향을받는된다 VO 2MAX의 70 %에서 75 km의 자전거 시합 후 즉시 1.5 시간 및 21 시간의 감소. * 운동 전 다른 표시 (쌍 t-test를 사후 분석 반복 측정 ANOVA, 적절한 경우, N = 19, P &lt;0.05). 값은 평균 ±에게 있습니다 표준 오류입니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54264/54264fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img고도 = &quot;그림 3&quot;SRC = &quot;/ 파일 / ftp_upload / 54264 / 54264fig3.jpg&quot;/&gt; . 그림 3 : 과립구 탐식은 과체중 / 비만 여성에서 전신 염증과 상관 관계가 과립구의 식균 작용은 혈중 C- 반응성 단백질, 전신 염증의 널리 인정 바이오 마커 (피어슨 제품 순간 상관 관계를 상관 관계; N = 87, R = 0.44; P &lt;0.01). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54264/54264fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

인간의 혈액에서 측정 과립구와 단핵구 식균 작용 및 산화 버스트 활동

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

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14.0K 조회수.  Appalachian State University. The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

비디오: Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

<em&gt; 체외</em인간 대식 세포의 분화와 양극화의 이질성을 연구&gt; 모델

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

연구

면역학 및 감염병학

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

인간 백혈구 및 혈소판의 분리 및 평가를위한 프로토콜 : 생물 에너지와 산화 버스트

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

이미지 기반 유동 세포 계측법 기술은 과립구 기능의 변화를 평가하기<em&gt; 체외</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

마우스 대 식 세포에 의해 식 균 작용의 시간 경과 3D 영상

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

헤모필루스 인플루엔자에 대한 호흡기 면역 반응 평가

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Monocyte-Macrophage Assay를 이용한 적혈구 항체 유의성 예측(Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay)

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

IgG-Opsonized 적혈구의 대식세포 식세포작용(Macrophage Phagocytosis)의 실시간 이미징

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

Source: Hartung, S., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60397">Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a method for quantifying the phagocytosis of FITC-labeled pathogenic fungal spores by human leukocytes. Phagocytosed spores are discerned from cell-adherent spores through counterstaining, followed by flow cytometric analysis.

인간 식세포에 의한 Fungal Spore Phagocytosis의 정량적 유세포 분석 측정

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Phagocytosis Assay

	Incubate 2 x ...

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Source: Meaney, M. P. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54264">Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood.</a> J. Vis. Exp. (2016)
This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes(과립구 및 단핵구에서 식세포작용 및 산화 버스트 활성을 평가하기 위한 분석)

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...