JB는 자연 과학 및 공학 연구위원회 (NSERC)에서 studentships에 의해 지원됩니다. 이 프로젝트에 대한 지원은 그랜트가 걸레 - 67066, 건강 연구의 캐나다 연구소에 의해 제공되었습니다.

이 프로토콜은 루츠 외에서 맞게되었습니다 1. 수확 및 골수 처리 <ol><li> 발목 부분의 경골과 대퇴골의 절연 :이 기증자의 마우스를 안락사와 70 % 에탄올로 다리를 스프레이. 무딘 집게로 단단히 최초의 발목을 파악하고 피부와 발목 부분의 경골을 폭로 기본 근육을 떼어 버리기 시작합니다. 뼈에 손상을 방지하려면, 무릎 관절을 그대로두고, 발목 부분의 경골에 천천히 및 병렬 컷. 대퇴골을 청소하려면, 무딘 포셉과 욕심에 의해 무릎 관절을 고정. 날카로운 가위와 곡선 포셉 한 쌍의으로 근육을 떨어져 청소하십시오. 엉덩이 관절이 노출되고 가위는 대퇴골의 머리와 엉덩이 관절 사이에 위치 수있을 때까지 위쪽으로 계속합니다. 인산염에 대퇴골과 고관절 관절과 장소 사이에 절단하여 뼈를 제거하면 얼음 호수 (PBS)를 버퍼. </li><li> 골수의 제거 : 가위와 곡선 집게를 사용하여 뼈에서 청소 남은 근육. 각 뼈의 epiphyses를 차단하고 중앙 구멍을 찾습니다. PBS로 로드된 10mL 주사기 및 25G0.5 &quot;바늘을 사용하지 않는 조직 코팅 페트리 접시에 골수를 플러시. </li><li> 골수의 처리 사항 : (모션 근근이 살아가고 아니라를 위아래 사용) 단일 세포 현탁액에 골수를 떼어 놓다에 1mL 주사기의 플런저의 고무 엔드를 사용합니다. 원뿔 팰컨 튜브의 골수를 수집하고, PBS로 잔여 골수를 씻어. </li></ol> 돌기 세포의 문화 <ol><li> hemocytometer에서 DC 언론과 카운트의 DC 엽 성의 전구 물질에 Resuspend 세포 (그림 1). 당신은 다양한 크기의 세포를 볼 수 있으며, DC의 엽 성의 전구 물질은 가장 크고 밝은 있습니다. Differentially 이러한 세포를 계산합니다. 이 대퇴골과 야생 타입 C57Bl / 6 마우스 (이전 6~8주)의 두 tibias에서, 당신은 25-40 X 10 6 총 직류 엽 성의 전구 물질 사이에 기대한다. <img src="/files/ftp_upload/old/769_2008_6_30_3/article769_figure_1.jpg" alt="" width="218" height="217" /> 그림 1 </li><li> 2 X 10 5 엽 성의 전구 물질의 DC / 40 NG / ML 재조합 murine GM - CSF와 보충 DC 미디어 ML의 밀도에 문화 세포. 비 - 조직 - 코팅 폴리스티렌 배양 접시에서 플레이트 세포. </li></ol> 미디어 (3 일) 추가 미디어를 새로 고치려면, 40 NG / ML GM - CSF와 보충 신선한 미디어의 총 볼륨의 절반을 추가합니다. 하나 미디어의 세번째 (6 일)과 성숙을 교체 미디어를 새로 고치려면 신중하게 미디어의 총 볼륨의 3 분의 1을 제거하고 문화 6 일 40 NG / ML GM - CSF와 보충 신선한 DC 미디어와 함께 볼륨을 교체하십시오. 원하는 경우, 돌기 세포는 크린 시토킨 또는 수신자 같은 수용체의 리간드를 사용하여 성숙을 위해 자극 수 있습니다. 동영상에서 DC가 5 NG / ML CPG와 하룻밤 자극에 의​​해 성숙되었다. 돌기 세포의 수확 DC 문화는 (그림 2) 완료됩니다. 세포 현탁액에서 느슨하게 번호판을 준수 둘 것입니다. 준수 세포는 조직 문화 스크레이퍼와 함께 요리 근근이 살아가고하고 PBS로 rinsing하여 제거할 수 있습니다. 세포의 총수는 주일 동안 문화와 차별화 기간 동안 5-8 배 증가합니다, 따라서 1-1.6 X 10 6 세포 / ML을 수확 예정입니다. <img src="/files/ftp_upload/old/769_2008_6_30_5/article769_figure_2.jpg" alt="그림 2" width="407" height="358" /> 그림 2 시약 <ol><li> DC 미디어 <ol><li> 와 보충 RPMI - 1640 미디어 : </li><li> 10% 태아 소 혈청 </li><li> 100 IU / ML 페니실린 </li><li> 100 μg / ML 스트렙토 마이신 </li><li> 1퍼센트 L - 글루타민 </li><li> 0.1 % 2 - 메르 캅 토 에탄올 </li><li> 1X 아닌 필수 아미노산 </li><li> 1X 나트륨 pyruvate </li></ol></li><li> GM - CSF 재조합 murine <ol><li> rmGM - CSF (Peprotech 병원, 뉴저지, 카탈로그 번호. 315-03) </li></ol></li></ol>

<ol>
	<li>Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., Schuler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+advanced+culture+method+for+generating+large+quantities+of+highly+pure+dendritic+cells+from+mouse+bone+marrow.">An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.</a> J. Immunol. Methods. 223, 77-92 (1999).</li><li>Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC-based+cancer+vaccines.">DC-based cancer vaccines.</a> J. Clin. Invest. 117, 1195-1203 (2007).</li></ol>

DC가이 타고난과 적응 면역의 상호 작용 연구에 유용하며, 백신 벡터로 고용하실 수 있습니다. 이 비디오에서는, 우리는 체외에서 골수를 분리하고 골수양 돌기 세포를 구별하는 단계를 증명하고있다. 문화 시대에 따라, 이들 세포는 미세 모두 자기편으로 자주 dendrites을 가지고 세포, 및 비 점착 성의 원형 세포를 시각하실 수 있습니다. DC가가 더욱 항원과 pulsing에 의해 항원 프레 젠 테이션에 대한 조작이나 크린...

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<td>recombinant murine GM-CSF</td>
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culture of myeloid dendritic cells from bone marrow precursors

골수양 돌기 세포 (DCS)은 자주 타고난과 적응 면역 메커니즘과 감염에 대한 초기 반응 사이의 상호 작용을 연구하는 데 사용됩니다. 이들은 세포를 제시 가장 강력한 항원 때문에 DC가 점점 항원 특정 면역 반응의 유도를 연구하기위한 백신 벡터로 사용되고 있습니다. 이 비디오에서는, 우리는 기증자 마우스 tibias과 대퇴골을 수확 골수을 처리하고 체외에서 DC가을 차별에 대한 절차를 설명합니다. 자극을 다음과 DC가 변화의 특성 : DC가 성숙은 CD4와 항원 프레 젠 테이션과 상호 작용 +와 CD8 + T 세포 수있는 반면 미숙 돌기 세포는 강력한 phagocytes 있습니다. 기능 활동이 변화는 세포 표면 마커 및 시토킨 생산의 upregulation과 일치. 많은 대리인은 크린 시토킨 및 수신자와 같은 수용체 리간드를 포함하여 성숙 DC가,하는 데 사용할 수 있습니다. 이 비디오에서는, 우리는 CPG 또는 모의 치료 하룻밤 자극을 다음과 같은 두 개의 공동 stimulatory 분자, CD86과 CD40, 그리고 시토킨, IL - 12의 표현의 흐름 cytometric 비교 보여줍니다. 차별화 후, DC가가 더욱 백신 벡터로 사용하기 위해 조작하거나 체외에 의해 항원 특정 면역 반응을 생성하는 펩티드 또는 단백질을 사용하여 pulsing, 또는 재조합 바이러스 벡터를 사용하여 transduced.

Watch this Scientific Journal Video about Culture of myeloid dendritic cells from bone marrow precursors at JoVE.com

Culture of myeloid dendritic cells from bone marrow precursors

이 동영상은 마우스 골수에서 골수양 돌기 세포를 차별에 대한 절차를 설명합니다. 마우스 경골과 대퇴골, 그리고 골수 처리의 분리가 증명하고 있습니다. 세포 표현형 및 CPG를 사용하여 IL - 12 생산 다음과 성숙을 묘사한 분화 전후의 세포 형태를 보여주는 사진, 그림이 표시됩니다.

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 ...

culture-of-myeloid-dendritic-cells-from-bone-marrow-precursors

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Biology

22.4K 조회수.  McMaster University. 이 동영상은 마우스 골수에서 골수양 돌기 세포를 차별에 대한 절차를 설명합니다. 마우스 경골과 대퇴골, 그리고 골수 처리의 분리가 증명하고 있습니다. 세포 표현형 및 CPG를 사용하여 IL - 12 생산 다음과 성숙을 묘사한 분화 전후의 세포 형태를 보여주는 사진, 그림이 표시됩니다.

비디오: Culture of myeloid dendritic cells from bone marrow precursors

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

냉동 세포에서 시작 문화 : 인간 ES 세포

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

연구

생물학

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

마우스 신경 줄기 세포 엽 성의 전구 물질의 문화

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

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% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

골수에 조혈 세포의 유도

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

마우스 골수에서 파골 세포의 유도

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Murine 골 수 유래 수지상 세포와 대 식 세포에 형광 성 융해 단백질의 표정

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.