저자는 신문을 읽고에 대 한 박사 P. Karagiannis와 그의 기술 지원에 대 한 교수 T. 호요 인정 하 고 싶습니다.

여기에 설명 된 모든 방법 로컬 윤리 위원회에 의해 승인 되었습니다 (도시샤 대학 윤리 위원회: 15033), 헬싱키의 선언에 의해 설정 된 표준 규격을 따라에서 했다. 동의 모든 참가자 로부터 얻은 것입니다. 참가자 들은 남성 레크리에이션 선수 (20.1 ± 1.2 년, 나이, 체중, 69.7 ± 6.2 k g, 높이, 176.1 ± 5.8 cm; 신체 지방 13.6 ± 1.5% 및 VO2최대 52.6 ± 6.6 mL/min1/kg1).
1입니다. 운동 프로토콜의 설립
참고: 현재 연구는 HIIE 프로토콜 중 antecubital 정 맥에서 혈액 샘플링에 의해 혈액 호르몬 및 대사 산물 역학의 평가의 구성 되어 있습니다.
<ol><li>낮은 강도에서 측에서 자전거의 5 분와 따뜻한을 수행을 요구 하십시오. 워밍업, 후 5 분 동안 휴식 하는 주제를 보자.</li>
	<li>다리 주기 측에서 다음 HIIE를 수행 하기 위해 주제를 물어: 30 s 높은 강도 운동, 구분 하 여 나머지 (그림 1)의 4 분의 3 관찰. 참고: 30 s 복싱 설정 되어야 합니다 강도 평등에서 90%로 이전 수행된 Wingate 테스트의 전원 평균. 다른 곳에 대 한 완전 한 윈 게이트 테스트8 참조.</li>
	<li>30 s 복싱에 대 한 작업 부하를 계산 하려면 다음 수식: 일 = 힘 x 거리 (식 1) 힘 = 질량 x 가속도 (식 2) 일 = (질량 x 가속도) 거리 (식 3) x 참고:이 모델에서는, 질량은 작업 부하 (kg), 그리고 가속도 9.81 m/s2이다. 거리는 바퀴 직경 (1.622 m). 때문에 바퀴 회전 3.7 타임된 /peal 회전, 페달 회전 당 거리 3.7 x 1.662입니다. 또한, 페달 주파수 90 레 브/분1이므로 우리 90 한 페달 거리를 곱하면 해야. 일 = 9.81 x 3.7 x 1.622 x 90 (식 4) x 작업 부하 작업 부하 = 일 / (9.81 x 3.7 x 1.622 x 90) (식 5)</li>
</ol>2입니다. 도관 법
참고: 튜브 및 vacutainers 표시 되어야 합니다 시간 손실을 방지 하려면 미리.
<ol><li>지 혈 대는 도관 법에 대 한 선택 하는 팔을 감싸 줍니다. 이렇게 하려면 선택한 팔 뒷면 신축성 밴드 놓습니다. 스트레칭 밴드, 팔 주위 밴드와 함께 루프를 확인 하 고 탄성을 넥타이.</li>
	<li>보이는 antecubital 정 맥을 선택 하 고 에탄올 또는 알코올 피부를 소독.</li>
	<li>피부와 15 °의 대략적인 각도에서 정 맥으로 멸 균된 정 맥의 끝을 삽입 합니다. 참고:은 정 원심 방향에 인접에서 삽입 한다. Cannulation 성공 하면 혈액의 backflush 관찰 한다.</li>
	<li>손으로 정을 장악 하 고 바늘을 부드럽게 제거 합니다. 참고: 운동을 하는 동안 바늘의 각도 유지, 면 조직 피부와 바늘 사이 사용할 수 있습니다. 테이프와 함께이 구조를 수정 합니다.</li>
	<li>헤 파 린과 첫 번째 혈액 샘플 (약 2.5 mL) aspirate는 vacutainer를 연결 합니다. 직후, 40 분 동안 얼음 상자에 넣어 혈액 샘플을 냉동. 혈액 가스 분석 하려고 하는 경우 보조 도움 요청.</li>
	<li>카 테 터에 튜브를 연결 하 고 혈액 응고를 피하기 위해 튜브의 전체 길이에 헤 파 린 주사. 나비를 닫습니다.</li>
	<li>테이프를 사용 하 여 카 테 터를 고정 시켜야 합니다. 이 시점에서 확인, 피사체의 팔 이동성 혈액 컬렉션에 방해가 되지 않습니다. 이 상태를 확인 하려면 반드시 바늘 위치 어떤 운동에 의해 영향을 받지 않습니다.</li>
</ol>3. 혈액 Samplings
<ol><li>때까지 테에만 피는 vacutainer를 사용 하 여는 헤 파 린을 제거 합니다. 혈액 플래쉬 백을 관찰 하는 경우 2.5 단계에 설명 된 대로 샘플을 수집 합니다.</li>
	<li>샘플 수집 후 혈액 응고를 피하기 위해 카 테 테 르 튜브에 헤 파 린을 다시 삽입할. 이 작업 후 단계 2.5 여러 샘플 컬렉션 반복 수 있습니다.</li>
	<li>마지막 샘플 수집 후 조심 스럽게 cannulation 튜브를 제거 하 고 알코올 피부를 청소. 가능 출혈을 방지 하려면는 catheterization 되었다 장소에 소독된 면 계속 주제 요청.</li>
	<li>혈액 vacutainer 튜브에 수집 후 신중 하 게 1.5 mL 튜브에 혈액의 약 1.5 mL를 통과 하 고 얼음에 튜브를 유지.</li>
	<li>젖 산 검출기를 사용 하 여 나머지 혈액에서 측정 혈액 젖 산. 같은 날에는 플라즈마 혈액 샘플을 (혈액의 약 1.5 mL) 구하십시오.</li>
	<li>플라스마 분리, 혈액 샘플을 4 oC에서 5000 x g에 5 분 동안 원심 및 새로운 1.5 mL 튜브에 상쾌한 분리.</li>
	<li>한 번 더 원심 분리를 반복 하 여 플라즈마 정화.</li>
	<li>혈장 분리 후 호르몬 분석까지-80 oC에서 샘플을 저장 합니다.</li>
	<li>ELISA 키트로 제조업체의 당 ( 재료의 표참조) 프로토콜을 사용 하 여 플라스마 인슐린을 측정 합니다.</li>
</ol>

<ol>
	<li>Wahl, P., Mathes, S., K&#246;hler, K., Achtzehn, S., Bloch, W., Mester, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+active+vs.+passive+recovery+during+Wingate-based+training+on+the+acute+hormonal,+metabolic+and+psychological+response.">Effects of active vs. passive recovery during Wingate-based training on the acute hormonal, metabolic and psychological response.</a> Growth Horm IGF Res. 23 (6), 201-208 (2013).</li><li>Wahl, P., Mathes, S., Achtzehn, S., Bloch, W., Mester, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+vs.+passive+recovery+during+high-intensity+training+influences+hormonal+response.">Active vs. passive recovery during high-intensity training influences hormonal response.</a> Int J Sports Med. 35 (7), 583-589 (2014).</li><li>Nalbandian, H. M., Radak, Z., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+active+recovery+during+interval+training+on+plasma+catecholamines+and+insulin.">Effects of active recovery during interval training on plasma catecholamines and insulin.</a> J Sports Med Phys Fitness. , (2017).</li><li>Stolen, T., Chamari, K., Castagna, C., Wislff, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+Soccer.">Physiology of Soccer.</a> Sport Med. 35 (6), 501-536 (2005).</li><li>Bangsbo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+soccer--with+special+reference+to+intense+intermittent+exercise.">The physiology of soccer--with special reference to intense intermittent exercise.</a> Acta Physiol Scand Suppl. 619, 1-155 (1994).</li><li>MacInnis, M. J., Gibala, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+adaptations+to+interval+training+and+the+role+of+exercise+intensity.">Physiological adaptations to interval training and the role of exercise intensity.</a> J Physiol. 595 (9), 2915-2930 (2017).</li><li>Emberts, T., Porcari, J., Dobers-Tein, S., Steffen, J., Foster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+intensity+and+energy+expenditure+of+a+tabata+workout.">Exercise intensity and energy expenditure of a tabata workout.</a> J Sports Sci Med. 12 (3), 612-613 (2013).</li><li>Smith, J. C., Hill, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+energy+systems+during+a+Wingate+power+test.">Contribution of energy systems during a Wingate power test.</a> Br J Sports Med. 25 (4), 196-199 (1991).</li><li>Dotan, R., Bar-Or, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Load+optimization+for+the+Wingate+Anaerobic+Test.">Load optimization for the Wingate Anaerobic Test.</a> Eur J Appl Physiol Occup Physiol. 51 (3), 409-417 (1983).</li><li>Carrasco-Benso, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+tissue+expresses+intrinsic+circadian+rhythm+in+insulin+sensitivity.">Human adipose tissue expresses intrinsic circadian rhythm in insulin sensitivity.</a> FASEB J. 30 (9), 3117-3123 (2016).</li><li>Hatfield, D. L., Nicoll, J. X., Kraemer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Circadian+Rhythm+on+Power,+Force,+and+Hormonal+Response+in+Young+Men.">Effects of Circadian Rhythm on Power, Force, and Hormonal Response in Young Men.</a> J Strength Cond Res. 30 (3), 725-732 (2016).</li></ol>

저자는 공개 없다.

이 연구에서는 antecubital 정 맥 catheterized 했다, 그리고 높은 간격 운동 중 혈액 샘플 수집 성공적으로 되었습니다. 이 방법론의 사용의 예를 들어, 혈액 샘플 플라스마 인슐린에 대 한 혈액 젖 산에 대 한 전후 (플라즈마 분리) 분석 되었다. 이 방법의 주요 장점은 수 혈액 샘플의 컬렉션 짧은 기간입니다. 동시에 그것은 몇 가지 한계는. 예를 들어, 제한 된 운동 양식 수 있는 그것의 사용; 조교 수, 고 ...

신체 활동 중 대사 및 endocrinal 마커의 측정 다른 운동 modalities의 생리 적인 의미를 이해 하는 관련성입니다. 일부 운동 형식 (예: 높은 강도 간격 운동 (HIIE)), 동안 혈액 대사와 호르몬 수준을 상대적으로 짧은 시간1,2,3변동.
구분 하 여 낮은 강렬한 운동 및 수동 기간4,5강도 높은 운동 짧은 기간 (즉, 축구, 농구, 럭비) 많은 스포츠에 의하여 이루어져 있다. HIIE 상기 스포츠와 유사성을 선물 하 고 따라서 선수6을 위한 훈련 방법으로 널리 사용. 또한, 스포츠와의 유사성 때문에 HIIE 사용 되었습니다 모델 운동 생리학7공부 하. 혈액 샘플링은 중요 한 생리 적 변수를 측정 하 고 그것은 자주 연구 목적 및 건강 상태 평가 대 한 사용. 그러나, HIIE 동안 혈액 샘플링 샘플 컬렉션 사이의 짧은 시간 때문에 기술적으로 어려운 수 있습니다. 이 문제에 해결책으로 여기 선물이를 받는 방법을 혈액 샘플 antecubital 정 맥 도관 법에 의해 짧은 기간 동안 과목 수행 HIIE.

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cycle ergometer</td>
			<td style="width:71px;">Monark</td>
			<td colspan="2" style="width:339px;">MONARK Ergomedic 874 E</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Catheter</td>
			<td style="width:71px;">Terumo</td>
			<td style="width:172px;">SR-FF2032</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vacutainer</td>
			<td style="width:71px;">Terumo</td>
			<td>PZ-D03</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Tube for catheter</td>
			<td style="width:71px;">Terumo</td>
			<td style="width:172px;">SP-PTW30L02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin</td>
			<td style="width:71px;">Terumo</td>
			<td>PF-10HF10UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactate pro2</td>
			<td style="width:71px;">Arkray</td>
			<td>&#65324;&#65332;-1730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin ELISA kit</td>
			<td>Abnova</td>
			<td>ELISA Kit KA0921</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of blood lactate and plasma insulin during high-intensity exercise by antecubital vein catheterization

신체 활동 중 대사 및 endocrinal 마커의 측정 다른 운동 modalities의 생리 적인 의미를 이해 하는 관련성입니다. 일부 동안 형식 (예: 높은 강도 간격 운동) 운동, 혈액 대사와 호르몬 수준을 짧은 기간 변경. 현재 연구에서 우리는 운동 하는 동안 몇 가지 혈액 샘플의 수집 수 있는 antecubital 정 맥 catheterize 하는 방법을 설명 합니다. 인슐린 및 정 맥 젖 산 농도 강도 높은 운동을 하는 동안 설명된 방법의 응용 프로그램에 의해 측정 되었다. 높은 강도 운동 구분 복구의 4 분의 3 30의 복싱 운동에 의하여 이루어져 있다. 마지막 복구 기간 후 윈 게이트 테스트 수행 되었다. Antecubital 정 맥에서 혈액 샘플 전에 각 30 s 한판 승부 후 전에 그리고 Wingate 테스트 후 얻은 했다. 그 결과, 운동을 하는 동안 플라스마 인슐린과 정 맥 혈액 젖 산 변화를 평가 가능 했다.

대표적인 결과로 우리 혈액 젖 산 및 인슐린 유사 6 과목에서 주기 측에 HIIE 동안 측정 하이 프로토콜을 사용. 피크 전력 및 Wingate 테스트에 대 한 평균 전력 732 ± 120와 549 ± 84 와트, 각각 있었다. 평균 전력은 HIIE의 30 s 발작 동안 전원 출력 (494 ± 75 와트)를 계산 하기 위해 사용 되었다.
그림 2 플라즈마 인?...

Watch this Scientific Journal Video about Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization at JoVE.com

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

여기 우리는 높은 강도 간격 훈련 기간 동안 antecubital 정 맥에서 혈액 샘플을 몇 가지를 얻기 위해 프로토콜을 제시. 이 프로토콜 혈액 대사 산물의 측정에 유용 수 있습니다 하 고 동안 endocrinal 마커 운동.

Antecubital 정 맥 도관 법에 의해 높은 강도 운동 중 혈액 젖 산 염과 플라스마 인슐린의 평가

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different ...

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Immunology and Infection

6.4K 조회수.  Kyoto University. 여기 우리는 높은 강도 간격 훈련 기간 동안 antecubital 정 맥에서 혈액 샘플을 몇 가지를 얻기 위해 프로토콜을 제시. 이 프로토콜 혈액 대사 산물의 측정에 유용 수 있습니다 하 고 동안 endocrinal 마커 운동.

비디오: Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

보완 고갈에 의해 특정 세포 집단의 고갈

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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면역학 및 감염병학

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

운동 유발성 Apoptosis 림프구의 결정을위한 손가락 - 스틱 혈액 샘플링 방법론

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokinetic 트랙 운동성 분석에 의한 세포 마이그레이션의 수량 평가

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

혈액 처리를위한 빠른 방법은 인간의 혈액에서 혈장 펩타이드 수준의 수율을 증가하는

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

형광 상관 분광학에 의해 측정 된 차 림프구의 플라즈마 막 분자 확산

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

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 ...