Name: generation of human monocyte-derived dendritic cells from whole blood
Uploaded: 2016-12-24T14:00:00
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 ?...

<ol>
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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.

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	<tbody>
		<tr>
			<td>APC Mouse Anti-Human CD19&#160; Clone&#160; HIB19</td>
			<td>BD Biosciences</td>
			<td>555415</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD83&#160; Clone&#160; HB15e</td>
			<td>BD Biosciences</td>
			<td>551073</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Imag Anti-Human CD14 Magnetic Particles&#160;</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&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10mL Serological Pipet</td>
			<td>Corning</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 25mL Serological Pipet</td>
			<td>Corning</td>
			<td>357525</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL 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&#160; Clone&#160; 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)&#160;</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&#160; Clone&#160; M5E2</td>
			<td>BD Biosciences</td>
			<td>555398</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD209&#160; Clone&#160; 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 0.5M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
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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 ...

The overall goal of this CD14 magnetic beads separation technique is to isolate monocytes from the blood for their further differentiation into dendritic cells or macrophages in vitro. This method can help answer key questions in the field of immunology about the recognition, processing, and presentation of pathogens or antigens by dendritic cells. The main advantage of this isolation procedure is that the 98 to 99%pure monocyte population can be obtained.Demonstrating the procedure will be Karolin Thurnes, a technician from our work group. Begin by splitting the blood pack volume among sterile 50 milliliter centrifuge tubes according to the amount of blood received. Adjust the final volume in each tube to 50 milliliters with sterile Dulbecco's PBS as necessary and centrifuge the samples.Using a sterile one milliliter serological pipette connected to a laboratory vacuum pump, remove the plasma and platelet layers leaving the boundary layers undisturbed. Then, use a new 25 milliliter pipette to combine the mononuclear cell boundary layers from two of the tubes in the new sterile 50 milliliter conical tube. Next, add 15 milliliters of density gradient medium to each of 450 milliliter conical tubes and pipetting slowly and carefully, overlay 25 milliliters of pooled blood cells onto the density gradient in each tube.Separate the cells by centrifugation. Then aspirate the plasma and platelet upper layers and transfer the PBMC interphases into new individual 50 milliliter conical tubes. Wash the PBMCs two times with up to 50 milliliters of sterile DPBS, pulling the cells after the first centrifugation and resuspending the pellet in 50 milliliters of fresh DPBS after the second.After counting, collect the cells by centrifugation for monocyte magnetic particle isolation. To purify the monocytes by magnetic particle isolation, adjust the PBMC concentration to eight times 10 to the 7th cells per milliliter of isolation buffer. Next, vortex the anti-human CD14 magnetic particles thoroughly and mix 50 microliters of particles for every one times 10 to the 7th PBMCs into the cells, and transfer the cell bead solution to a sterile 15 milliliter tube.After 30 minutes at room temperature, adjust the total volume to 12 milliliters of isolation buffer, and transfer two milliliters of the cell particle suspension into each of six sterile round bottom tubes. Immediately place the tubes onto the cell separation magnet for 10 minutes at room temperature. Then remove the supernatant containing the peripheral blood lymphocytes from each tube.Remove the tubes from the magnet and add two milliliters of fresh isolation buffer to each cell sample. Gently pipette up and down to resuspend the cells and particles, and return the tubes to the magnet for another five minutes. At the end of the incubation, remove the supernatant again, and resuspend the cells in two milliliters of fresh isolation buffer as just demonstrated.Then pull the CD14 positive monocyte containing cell fractions into a new 50 milliliter tube and bring the final volume of the tube up to 50 milliliters in sterile DPBS. To differentiate the purified monocytes into dendritic cells, collect the cells by centrifugaiton and resuspend the pellet in 50 milliliters of DPBS. After counting, spin down the cells again and resuspend the pellet at a one times 10 to the 6th cells per milliliter concentration and prewarmed culture medium.Transfer three milliliters of cells into each well of a six well plate and add recombinant human Interleukin four and GMCSF to each well for a 48 hour incubation at 37 degrees Celsius and 5%carbon dioxide. On the second day of culture, restimulate the cells with fresh cytokines and return the plate to the incubator. On day five, pull the supernatants and gently pipette fresh medium into the wells several times to harvest the immature dendritic cells.After density gradient separation, a typical PBMC fraction is comprised of about 4%B-lymphocytes, 28%monocytes and 68%other cells. 44%of which are t-lymphocytes. After magnetic particle separation, flow cytometric analysis of the negative fraction reveals a similar percentage of b-lymphocytes, a higher percentage of the other cell populations and a greatly reduced percentage of monocytes.Characterization of the positive fraction demonstrates the high purificaiton efficiency with over 99%of the cells expressing CD14. After five days of Interleukin four and GMCSF simulation, the CD14 positive cells differentiate into immature CD83 negative DC-SIGN positive monocyte derived dendritic cells which are comparable to dermal dendritic cells in the morphology, behavior, and receptor expression. Once mastered, this technique can be completed in four to five hours if it is performed properly.After watching this video, you should have a good understading of how to isolate monocytes from whole blood and differentiate them into dendritic cells. This technique is very important for researchers in the field of immunology to explore not only dendritic cell, but also monocyte and macrophage function in the primary in vitro human cell system. Following this procedure, other methods like microscopic analysis, flow cytometry, infection or antigen presentation essays can be performed to answer questions about dendritic cell biology, function and interactions with antigens.Don't forget that working with blood can be hazardous and that precautions such as wearing gloves, a lab coat or having a hepatitis vaccination should always be taken before beginning this procedure.

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Research

JoVE Journal

Immunology and Infection

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

연구

면역학 및 감염병학

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

활성화 및 인간 단핵구 유래 수지상 세포에서 IL-1β를 사용하여 NLRP3 inflammasome을 활동의 측정

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

대 식세포 콜로니 자극 인자 차별화 된 인간 단핵구 유래 대 식세포의 문화

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

siRNA 전기 포진 포진 심플렉스 바이러스 유형 1-감염 단핵구 파생 수지상 세포에서 자가 포식을 조절

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

인간 단핵구 유래 대식세포에서 근접성을 유도한 염증성 카스파제의 시각화

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

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 ...