Name: generation of human monocyte-derived dendritic cells from whole blood
Uploaded: 2016-12-24T14:00:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Generation of Human Monocyte-derived Dendritic Cells from Whole Blood at JoVE.com

We would like to thank our technician Karolin Thurnes, Divison of Hygiene and Medical Microbiology, and Dr. Annelies M&#252;hlbacher and Dr. Paul H&#246;rtnagl, Central Institute for Blood Transfusion and Immunological Department, for their valuable help and support regarding this manuscript. We thank the Austrian Science Fund for supporting this work (P24598 to DW, P25389 to WP).

윤리 문 : 동의 글은 수혈 및 면역 부서, 오스트리아 인스 브루 크의 중앙 연구소 참여하는 모든 혈액 기증자로부터 얻은 것입니다. 과학적 목적을 위해 익명 남은 표본의 사용은 인스 브루 크의 의과 대학의 윤리위원회에 의해 승인되었다. 말초 혈액 단핵 세포의 1. 농축 (PBMC를) <ol><li> 원심 분리하여 PBMC를의 농도. <ol><li> 수신 된 혈액의 양에 따라 멸균 50 ㎖ 원심 분리 튜브에 혈액 팩 분할 혈액을 연다. </li><li> 필요한 경우, 멸균 둘 베코 인산염 완충 식염수 (D-PBS)와 각 튜브에 대한 50 ㎖로 볼륨을 조절합니다. </li><li> 원심 분리기에 튜브를 삽입하고, 브레이크없이, 실온 (RT)에서 15 분 동안 400 × g으로 그들을 회전. </li><li> boundar두고 멸균 10 ㎖ 혈청 피펫을 사용하여 혈장과 혈소판을 포함하는 상층을 그린다Y 층 흐트러. 주 : FCS 대신의자가 혈장이 배지를 보충하기 위해 사용되는 경우, 플라즈마가 수집되어야하며,이 단계에서 열이 불 활성화. </li><li> 이 50 ㎖ 원심 분리기 튜브에서 단핵 세포 (경계층)를 수집하고 멸균 10 ㎖의 혈청 학적 피펫을 사용하여 하나의 멸균 50 ML 튜브로 전송할 수 있습니다. </li><li> 멸균 D-PBS를 사용하여 각 튜브 50ml로 볼륨을 조절합니다. </li><li> 원심 분리기에 튜브를 놓고 브레이크없이 실온에서 15 분 동안 400 XG에 그들을 스핀. </li><li> 교란 경계층을 떠나 멸균 10 ㎖ 혈청 피펫을 사용하여 혈장과 혈소판을 포함하는 상층을 그린다. </li><li> 50 ㎖의 원심 분리기 튜브에서 단핵 세포 (경계층)를 수집하고 멸균 10 ㎖의 혈청 학적 피펫을 사용하여 두 멸균 50 ㎖ 수집 튜브로 전송할 수 있습니다. </li><li> 멸균 D-PBS 각 튜브 50ml로 볼륨을 조절합니다. </li></ol></li><li> 분리밀도 구배 원심 분리 한 PBMC의 (도 1). <ol><li> 각 튜브에 밀도 구배 매질 네 50 ㎖ 튜브에 피펫 15ml의 준비. </li><li> 포집 관 (단계 1.1.10)에서 풀링 된 세포 / 혈액 25 mL를 취하여 조심스럽게 밀도 구배 매체를 오버레이. </li><li> 원심 분리기에 튜브를 삽입하고, 브레이크없이 RT에서 30 분 동안 700 × g으로 그들을 회전. </li></ol></li><li> 수집 및 PBMC를 정화. <ol><li> 멸균 10 ㎖ 혈청 피펫을 사용하여 혈장, 혈소판, 방해받지 PBMC 층을 떠나는 함유 상부층을 그린다. </li><li> 모든 튜브에서 PBMC의 계면를 수집하고 멸균 25 ㎖의 혈청 학적 피펫을 사용하여 두 멸균 50 ㎖ 튜브에 세포를 풀. </li><li> 멸균 D-PBS 각 튜브 50ml로 볼륨을 조절합니다. 원심 분리기에 튜브를 놓고 브레이크와 4 ° C에서 15 분 동안 400 XG에 그들을 스핀. </li><li> 뜨는을 대기음하고 다시 중단멸균 D-PBS에있는 세포. 두 튜브에서 세포를 수영장과 멸균 D-PBS로 50 ㎖로 볼륨을 조절합니다. </li><li> 원심 분리기에 튜브를 놓고 브레이크 4 ° C에서 10 분 동안 400 XG에 그들을 스핀. </li><li> 뜨는을 기음과 멸균 D-PBS의 세포를 다시 일시 중지합니다. D-PBS 450 μl를 포함하는 1.5 ML 튜브에 멸균 D-PBS 50 ml를 피펫 50 μL에 볼륨을 조정합니다. </li><li> 소용돌이 1.5 ML 튜브 적극적와 노이 바 우어 챔버 또는 다른 세포 계수 장비의 셀을 계산합니다. </li><li> 원심 분리기에 50 ㎖ 튜브를 놓고 브레이크 4 ° C에서 10 분 동안 400 XG에 그들을 스핀. </li></ol></li></ol> 반대로 인간 CD14 자성 입자에 의해 단핵구 2. 분리 <ol><li> 자성 입자와 PBMC를의 레이블. <ol><li> 차단 완충액을 사용하여 8 × 107 세포 / ml로 PBMC 농도를 조정한다. </li><li> 와동 항 인간 CD14 자성 입자 thoroughlY마다 1 × 10 7 PBMC를 위해 입자의 50 μl를 추가합니다. </li><li> 철저히 셀 입자 현탁액을 혼합하고 실온에서 30 분 동안이를 부화. </li></ol></li><li> 양극과 음극 분수의 분리. <ol><li> 절연 버퍼를 사용하여 12 ml의에 볼륨 업을 조정합니다. </li><li> 각 튜브에 세포 입자 현탁액의 여섯 멸균 둥근 바닥 튜브 및 피펫 2 ml의를 준비합니다. </li><li> 즉시 자석에 튜브를 배치합니다. RT에서 10 분 동안이를 부화. </li><li> 자석에 튜브를 유지하고 조심스럽게 상층 액을 그립니다. 상층 액은 부정적인 부분이 포함되어 있습니다. </li><li> 자석에서 튜브를 제거하고 분리 버퍼 2 ㎖를 추가합니다. </li><li> 부드럽게 피펫 팅 아래로 세포를 다시 일시 중지합니다. </li><li> 다시 자석에 튜브를 놓습니다. RT에서 5 분 동안 인큐베이션. </li><li> 자석에 튜브를 유지하고 조심스럽게 상층 액을 그립니다. 상층 액은 부정적인 부분이 포함되어 있습니다. </li><li>자석에서 튜브를 제거하고 각 튜브를 분리 버퍼 2 ㎖를 추가합니다. 부드럽게 피펫 팅 아래로 세포를 다시 일시 중지합니다. </li><li> 멸균 50 ML 튜브에 긍정적 인 부분을 전송하고 멸균 D-PBS를 사용하여 50ml로 볼륨을 조절합니다. </li></ol></li></ol> IL-4와 GM-CSF와 격리 된 단핵구의 3 자극 <ol><li> 원심 분리기에 튜브를 놓고 브레이크 4 ° C에서 10 분 동안 400 XG에 그것을 회전. </li><li> 대기음 뜨는 D-PBS 50 ㎖로 세포를 재 - 보류. </li><li> D-PBS 450 μl를 함유하는 1.5 ㎖의 튜브에 피펫 50 μL. 소용돌이 1.5 ML 튜브 적극적와 노이 바 우어 챔버 또는 다른 세포 계수 장비의 셀을 계산합니다. </li><li> 원심 분리기에 50 ML 튜브를 놓고 브레이크 4 ° C에서 10 분 동안 400 XG에 그것을 회전. 주 : 말초 혈액 림프구 (PBL들)를 함유하는 네가티브 분획, 또한 필요하면 세정을 수행하고 카운팅을 t 유사한 단계긍정적 인 부분의 호스 (3.1-3.5 단계). </li><li> 미리 예열 문화 매체 (RPMI 1640 10 % 열 - 불 활성화 FBS와 1 % L - 글루타민 용액을 보충 매체) 및 피펫 3 ㎖ / 잘 6 웰 플레이트에 1 × 106 / ㎖의 세포 농도를 조정합니다. 참고 : 열 불 활성화자가 플라즈마를 사용하는 경우, 실험 셋업에 따라, 1-5%자가 플라즈마로 10 % FCS를 교체합니다. </li><li> 배양액으로 직접 피펫 팅하여 사이토 카인의 재조합 인간 IL-4 (250 U / ml) 및 재조합 인간 GM-CSF (1,000 U / ㎖)으로 단핵구를 자극한다. 37 ° C에서 품어 5 % CO 2. </li><li> 배지에 직접 사이토킨 피펫 팅하여 48 시간 후 IL-4 (250 U / ml) 및 GM-CSF (1,000 U / ㎖)와 함께 세포를 재 자극한다. 주 : 배지의 pH를 지표로 나타낸 산성화의 흔적이 있다면, 예열 배양액 배지의 절반을 교체한다. </li><li> 수확을 위해 하루 5 수지상 세포를 미 숙다운 스트림 6 웰 플레이트에서 그리고 50 ml의 원심 분리기 튜브에 최대 배지를 피펫 팅 후 몇 번 아래로 배양 된 세포를 피펫 팅으로 실험. </li><li> 세포를 세어 CD11b를, 중 CD11c에 대한 항 인간 항체와 격리 된 단핵 세포의 샘플을 얼룩과 함께 세포 생존 염료와 DC-SIGN / CD209. 표면 오염시 항체의 비특이적 결합을 방지하기 위해, 시약 또는 소 혈청 알부민 (BSA) 버퍼를 차단 Fc 수용체의 사용이 추천된다. </li><li> 생성이 iDCs의 순도 및 생존력을 평가하기 위해 유세포 수행하여, 제조자의 프로토콜에 따라, 세포 생존 성 염료를 사용하여 샘플을 분석한다. </li></ol>

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The authors have nothing to disclose.

이 프로토콜은 자성 나노 입자 - 기반 분석법을 이용하여 항 - 응고 혈 단핵 세포에서 인간의 분리를 통하여 단핵 세포 - 유래 수지상 세포 (MDDCs)의 생성을 설명한다. 이 프로토콜에서, 원심 분리 단계는 PBMC의 분획의 농축에 이르는 세포 분리 과정 상류 행한다. 세포를 원심 분리 중에 손실되지만, 밀도 구배 배지 200-300 ml의 요구 밀도 구배 매체의 전혈 팩의 내용을 오버레이 때문에 비용 효율적으?...

수지상 세포 (DCS)는 우리의 면역 시스템의 중요한 전문 항원 제시 세포이다. 미성숙 수지상 세포 (이 iDCs)는 피부 또는 점막 조직에 상주하며 침입 병원체와 상호 작용하는 제 면역 세포 중에서 따라서이다. 그들은 병원체 검출 이후에 T-와 B 세포 반응을 활성화 할 수 있기 때문에 수지상 세포는 선천성 및 적응성 면역 체계 1 사이의 다리를 나타냅니다. 또한, 그들은 때문에 이러한 IL-1β, IL-6, IL-12 사이토 카인의 다량의 분비 염증성 면역 반응에 기여한다. 수지상 세포는 NK 세포의 활성화 및 주 화성에 의한 감염 부위에 다른 면역 세포를 끈다. 미성숙 수지상 세포는 수지상 세포 (이 iDCs) 및 형태와 기능에 기초하여 성숙 수지상 세포 (MDCS) 2로 나누어 질 수있다. 많은 패턴 인식 수용체의 하나 (예를 들어, 수신자 같은 수용체, C 형에 의한 외국 항원의 인식 후렉틴 또는 다량 세포 표면에 발현) 수용체를 보완이 iDCs는 큰 변화를 겪는 성숙한 시작한다. 항원 제시에 필수적인 분자가 3 상향 조정되는 반면이 성숙 과정에서, 항원 캡처 수용체는, 아래로 조절된다. 성숙한 DC가 주요 조직 적합성 복합체 I 및 II (MHC I과 II)를 상향 조절, T 림프구의 항원 제시 및 활성화에 필수적인 CD80 및 CD86와 같은 공동 자극 분자. 또한, 세포 표면의 케모카인 수용체 CCR7의 발현은 림프절 말초 조직에서 수지상 세포의 이동을 가능하게하는 유도된다. 마이그레이션은 림프절 4-6로 케모카인 리간드 19 (CCL19 / MIP-3B)과 케모카인 리간드 21 (CCL21 / SLC) 그라데이션을 따라 수지상의 &quot;구름&quot;에 의해 촉진된다. 마이그레이션 후, MDC에 따라서 INI, CD4 +와 CD8 + T 세포를 순진하기 위해 처리 된 항원을 제시침입 병원균 7에 대한 적응 면역 반응을 tiating. 림프절에서 T 세포와의 상호 작용이도 8 바이러스의 확산과 관련된다. 다른 시험관 연구는 DC가 효율적으로 캡처하고 활발한 감염 9-12이 전송 결과 T 세포 것을 HIV를 전송하는 것으로 나타났습니다. 이 실험은 림프절에 주변에서 셔틀로 그 생체 내 HIV의 공격의 수지상를 강조 표시합니다. 항원 제시 중에 DCS는 이펙터 T 헬퍼 세포의 분화를 형성하고, 따라서, 미생물에 대한 전체 면역 반응의 결과가이 상호 작용에서 매우 중요한 판단 인터루킨 분비. 별도로 1 형 (받은 Th1)과 제 2 형에서 (TH2) 이펙터 T 세포, CD4 + T 헬퍼 세포 (예를 들어, 유형 17 (Th17) 및 유형 22 (Th22) T 세포)에 설명 된 다른 부분 집합, 그들의 유도 기능은 충분히 조사하고있다. 수지상은 또한에 관여T 조절 세포 (Tregs) (13, 14)의 생성. 이 세포들은 면역 억제하고 중지하거나 또는 유도 이펙터 T 세포의 증식을 하향 조절함으로써 면역 관용 개발 중요하다 할 수있다. 인간 기존의 DC (CDCS)는 골수 기원 세포의 여러 하위 집합을 포함한다 (즉, 랑게르한스 세포 (LCS)과 피부 및 간질 DC가) 또는 림프 기원 (즉, 형질 DC가 (올라가고)). 시험 관내 실험 또는 DC 예방 접종 전략의 경우, 단핵구 유래 수지상 정기적으로 피부 수지상의 모델로 사용된다. 이 세포들은 기존의 골수 수지상 세포 생리학, 형태 및 기능의 유사성을 보여줍니다. 이들은 건강한 공여자로부터 단리 12,15-18 단핵구에 인터루킨 4 (IL-4), 과립구 대 식세포 콜로니 자극 인자 (GM-CSF)의 첨가에 의해 생성된다. 수지상 세포는 또한 직접적으로 피부 또는 점막 생검으로부터 분리, 또는 B 수 가능E는 CD34 + 전 자궁 획득 탯줄 혈액에서 분리 한 조혈 전구 세포에서 개발 하였다. 여기서는 단핵구 절연 및 밀도 구배 원심 분리에 의해 말초 혈액 단핵 세포 (PBMC) 농축 한 후 항 - 인간 혈액 응고 촉진하는 방법을 보여준다. 5 일간 배양 한 후, 특정 조건 하에서 인간 단핵 세포는이 iDCs로 분화하고 비 임상 설정에서 실험 절차에 대한 준비가되어 있습니다.

<table><tbody><tr><td>APC Mouse Anti-Human CD19 Clone HIB19</td><td>BD Biosciences</td><td>555415</td><td></td></tr><tr><td>APC Mouse Anti-Human CD83 Clone HB15e</td><td>BD Biosciences</td><td>551073</td><td></td></tr><tr><td>BD Imag Anti-Human CD14 Magnetic Particles</td><td>BD Biosciences</td><td>557769</td><td></td></tr><tr><td>BD Imagnet</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BSA (Albumin Fraction V)</td><td>Carl Roth</td><td>EG-Nr 2923225</td><td></td></tr><tr><td>Costar 6 Well Clear TC-Treated Multiple Well Plates</td><td>Costar</td><td>3506</td><td></td></tr><tr><td>Density gradient media: Ficoll-Paque Premium</td><td>GE Healthcare Bio-Sciences</td><td>17-5442-03</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Falcon 10&amp;nbsp;ml Serological Pipet</td><td>Corning</td><td>357551</td><td></td></tr><tr><td>Falcon 25&amp;nbsp;ml Serological Pipet</td><td>Corning</td><td>357525</td><td></td></tr><tr><td>Falcon 50&amp;nbsp;ml High Clarity PP Centrifuge Tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>Falcon Round-Bottom Tubes</td><td>Corning</td><td>352054</td><td></td></tr><tr><td>FITC Mouse Anti-Human CD3 Clone HIT3a</td><td>BD Biosciences</td><td>555339</td><td></td></tr><tr><td>Ghost Dye Violet 510 (Cell Viability Reagent)</td><td>Tonbo biosciences</td><td>13-0870</td><td></td></tr><tr><td>GM-CSF</td><td>MACS Miltenyi Biotec</td><td>130-093-862</td><td></td></tr><tr><td>Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America)</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>Hettich Rotanta 460R</td><td>Hettich</td><td>---</td><td></td></tr><tr><td>IL-4 CC</td><td>PromoKine</td><td>C-61401</td><td></td></tr><tr><td>Isolation buffer: BD IMag Buffer (10x)</td><td>BD Biosciences</td><td>552362</td><td></td></tr><tr><td>L-Glutamine solution</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Microcentrifuge tubes, 1.5 ml, SuperSpin</td><td>VWR</td><td>211-0015</td><td></td></tr><tr><td>PE Mouse Anti-Human CD14 Clone M5E2</td><td>BD Biosciences</td><td>555398</td><td></td></tr><tr><td>PE Mouse Anti-Human CD209 Clone DCN46</td><td>BD Biosciences</td><td>551265</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>Sigma-Aldrich</td><td>R0883</td><td></td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575020</td><td></td></tr></tbody></table>

generation of human monocyte-derived dendritic cells from whole blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

수 크로스 쿠션을 사용하는 항 응고 혈의 원심 분리 후, 말초 혈 단핵 세포 (PBMC)를 밀도 구배 배지 (도 1)의 상단에 계면에서 농축된다. PBMC를 전원이 그려진 후, FACS 분석은 혈통 마커를 사용하여 PBMC를 (예를 들어, CD3 T 림프구를 들어, CD14 단핵구, 그리고 CD19 B 림프구에 대한) 내에서 서로 다른 세포 집단의 특성을 수행한다. 도 2a는 수집 및 ?...

Watch this Scientific Journal Video about Generation of Human Monocyte-derived Dendritic Cells from Whole Blood at JoVE.com

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

전혈로부터 인간 단핵구 유래 수지상 세포의 생성

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

generation-of-human-monocyte-derived-dendritic-cells-from-whole-blood

Research

JoVE Journal

Immunology and Infection

20.1K 조회수.  Medical University of Innsbruck. Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

비디오: Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

골수 엽 성의 전구 물질에서 골수양 돌기 세포의 문화

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

연구

생물학

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

골수의 생성은 2 광자 영상에서 사용하기 위해 Murine 돌기 세포를 유래

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

면역학 및 감염병학

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

대사 표현형을 각기 다른 미성숙, 성숙 및 Tolerogenic 수지상 세포의 생성

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

이미징 유동 세포 계측법에 의해 인간 단핵구 유래 수지상 세포의 특성 : 두 단핵구 격리 프로토콜 사이의 비교

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

골수성 창시자 및 정렬 전략 소설 셀을 사용 하 여 Murine 골에서 수지상 세포 선구자의 큰 숫자의 생성

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

소 단핵구 유래 수지상 세포를 이용한 백신 면역원성 측정

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

Sialidase 처리를 사용하여 분리된 PBMC에서 탈-시알릴화된 단핵구 유래-DC

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

저자 스포트라이트: 세포 표면의 Sialidases-Based Enzymatic Treatment를 사용한 수지상 세포 성숙 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Generation of Monocyte-Derived Dendritic Cells with Differenting Sialylated Phenotypes (시알릴화 표현형이 다른 단핵구 유래 수지상 세포 생성)

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

전혈로부터 인간 단핵구 유래 수지상 세포의 생성

요약

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골수성 창시자 및 정렬 전략 소설 셀을 사용 하 여 Murine 골에서 수지상 세포 선구자의 큰 숫자의 생성

소 단핵구 유래 수지상 세포를 이용한 백신 면역원성 측정

Generation of Monocyte-Derived Dendritic Cells with Differenting Sialylated Phenotypes (시알릴화 표현형이 다른 단핵구 유래 수지상 세포 생성)

Generation of Monocyte-Derived Dendritic Cells with Differenting Sialylated Phenotypes (시알릴화 표현형이 다른 단핵구 유래 수지상 세포 생성)

Generation of Monocyte-Derived Dendritic Cells with Differenting Sialylated Phenotypes (시알릴화 표현형이 다른 단핵구 유래 수지상 세포 생성)