著者は、博士 p. Karagiannis ペーパーを読むと彼の技術的なサポートのための教授 t. ほようを認識したいと思います。

ここで説明したすべてのメソッドは、ローカル倫理委員会によって承認されている (同志社大学倫理委員会: 15033)、ヘルシンキ宣言によって設定された基準に厳密に従っていた。すべての参加者からインフォームド コンセントを得た。被験者は男性のレクリエーション スポーツ選手 (年齢、20.1 ± 1.2 年; 重量 69.7 ± 6.2 kg; 176.1 ± 高さ 5.8 cm; 体脂肪 13.6 ± 1.5% と VO2、最大 52.6 ± 6.6 mL/分1/kg1)。
1. 運動プロトコルの確立
注: 本研究は HIIE プロトコルの中に肘静脈から採血して血液ホルモンと代謝物の動態評価の成っていた。
<ol><li>低強度での自転車エルゴメータでのサイクリングの 5 分を暖かいを実行する対象を求めます。ウォーム アップの後、残り 5 分の件名をしましょう。</li>
	<li>下肢エルゴメータで次の HIIE を実行する対象を求める: 30 s 高強度運動、区切られた (図 1)、残りの 4 分の 3 の発作。 注: 30 s 発作べき強度と同額に設定 90% 以前に実行されたウィンゲート テストのパワー平均。ウィンゲート テストの完全な説明の8を他の場所で参照してください。</li>
	<li>30 s 発作の負荷を計算するには、次の数式を使用します。 仕事 = 力 x 距離 (関係式 1) 力 = 質量 x 加速度 (式 2) 仕事 = (質量 x 加速度) x 距離 (式 3) 注: このモデルは作業負荷 (kg) 質量と加速度は 9.81 m/s2です。距離は、車輪の直径 (1.622 m) です。3.7 時間/peal 回転車輪の回転するため、ペダル回転あたりの距離は 3.7 x 1.662 です。さらに、ペダル回転数は 90 回転/分190 の 1 つのペダル距離の乗算するあります。 仕事 = 9.81 x 3.7 x 1.622 x 90 (関係式 4) x 作業負荷 作業負荷 = 仕事/(9.81 x 3.7 x 1.622 x 90) (式 5)</li>
</ol>2. カテーテル
注: チューブと vacutainers ラベルを付けるように事前に時間を失うことを避けるために。
<ol><li>カテーテル検査の選択腕に駆血帯をラップします。これを行うには、選択した arm の背面にあるゴムバンドを配置します。伸縮バンド、腕の周りのバンドで輪を作るし、弾性を結ぶ。</li>
	<li>目に見える肘静脈を選択し、エタノールやアルコールで皮膚を消毒します。</li>
	<li>滅菌のカニューレの先端を皮膚に 15 ° のおおよその角度で静脈に挿入します。 注: から近位遠位方向にカニューレを挿入します。穿刺に成功した場合は、血液のバック フラッシュを観察すべき。</li>
	<li>手でカニューレを固定し、そっと針を削除します。 注: 運動中に針の角度を維持するために綿組織は皮膚と針の間に使用することがあります。この構造体をテープで固定します。</li>
	<li>ヘパリンと最初の血液サンプル (約 2.5 mL) の吸引、バキュテナーを接続します。すぐの後で、40 分のアイス ボックスにそれを置くことによって血液サンプル冷蔵庫で冷やします。血液ガス分析しようとしている場合は、アシスタントを助けを求めます。</li>
	<li>カテーテルにチューブを接続し、血液凝固を避けるために管の長さがフルにヘパリンを注入します。蝶を閉じます。</li>
	<li>テープを使用して、カテーテルを安定させます。この時点でことを確認、被写体の腕移動は血のコレクションと干渉しません。この条件を確認するため針の位置は、任意の動きの影響を受けないことを必ずください。</li>
</ol>3. 血液サンプリング
<ol><li>ヘパリンを削除するには、カテーテルに血液だけがあるまで、バキュテナーを使用します。血液のフラッシュ バックを観測すると、2.5 の手順で説明するようにサンプルを収集します。</li>
	<li>サンプル コレクション後血液凝固を避けるためにカテーテル チューブにヘパリンを再び挿入します。この操作の後ステップ 2.5 は、いくつかのサンプル コレクションの繰り返されるかもしれません。</li>
	<li>最後のサンプルが収集された後慎重にカニュレーション チューブを削除し、アルコールで皮膚をきれいします。可能な限り出血を避けるため、カテーテル検査が作られた場所で滅菌綿を保つために主題を求めます。</li>
	<li>バキュテナー チューブで収集された血液は、慎重に血液の約 1.5 mL を 1.5 mL チューブに渡すし、チューブを氷の上。</li>
	<li>測定血中乳酸乳酸検出器を用いた血の残りから。同じ日に、プラズマの採血 (血液の約 1.5 mL) を取得します。</li>
	<li>血漿分離 4 oC で 5000 × g で 5 分間の血液サンプルを遠心し、新しい 1.5 mL チューブに上清を分離します。</li>
	<li>プラズマを浄化するために遠心分離の 1 つのより多くの時間を繰り返します。</li>
	<li>血漿分離後のホルモンの分析まで-80 oC でサンプルを格納します。</li>
	<li>製造元のあたり (材料の表を参照) をプロトコルとして ELISA キットを使用してインスリンを測定します。</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>

著者が明らかに何もありません。

本研究では肘静脈が我々 だったと血液採取した高インターバル運動中に正常に。この手法の利用例として採血を行った (後に血漿分離) と血中乳酸の血漿インスリンのこの方法の主な利点は、血液サンプルのコレクション要するに期間できるということです。同時に、それはいくつかの制限があります。その使用を許可する限られた運動様式などアシスタントの数、必要し、専門訓練を受け?...

身体活動時代謝と内分泌マーカーの測定は、さまざまな運動療法の生理学的意味を理解するための関連性の高いです。いくつかの運動の様相 (例えば、高強度インターバル トレーニング (HIIE)) 中、血液代謝やホルモンのレベルは比較的短時間1,2,3で変動します。
(すなわちサッカー、バスケット ボール、ラグビー) の多くのスポーツは、低の激しい運動やパッシブ期間4,5で区切られた高強度運動の短い期間で構成されます。HIIE は前述のスポーツとの類似点を提示し、選手6トレーニング法として広く利用されています。さらに、スポーツとの類似性のため HIIE として使用されていますモデル7運動生理学を勉強します。研究目的および物理的な条件の評価に使用頻繁および採血生理学的変数を測定することが欠かせません。ただし、HIIE の中に採血はサンプル コレクション間の短い期間のため技術的に困難することができます。ここで、この問題の解決策として間を取得する血液サンプル肘静脈カテーテル法によって短い期間科目を実行 HIIE の方法を示す.

<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

身体活動時代謝と内分泌マーカーの測定は、さまざまな運動療法の生理学的意味を理解するための関連性の高いです。いくつかの中に運動療法 (例えば、高強度インターバル トレーニング)、血液代謝やホルモンのレベルを変更で短い期間。本研究では運動中にいくつかの血液サンプルのコレクションを可能にする肘静脈カテーテルを入れて下さい方法をについて説明します。記述法の適用によって高強度の運動中インスリンおよび静脈の乳酸濃度を測定しました。運動は、高強度運動回復の 4 分で区切られた 3 つの 30 s 発作から成っていた。最後の回復期間後ウィンゲート テストを行った。前に、各 30 秒試合後と前に、とウィンゲート テスト後、肘の静脈から血液サンプルが得られました。その結果、運動中に血漿インスリンと静脈血乳酸のバリエーションを評価することが可能だった。

代表的な結果としてエルゴで HIIE の中に 6 科目で血の乳酸およびインスリン変動を測定するのにこのプロトコルを使用しました。ピーク電力と平均電力ウィンゲート テスト 732 ± 120 と 549 ± 84 ワットであった。平均電力は、HIIE の 30 s 発作時に出力 (494 ± 75 ワット) を計算していました。
図 2は、血?...

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

肘静脈から高強度のインターバル トレーニング中にいくつかの血液サンプルを取得するためのプロトコルをご紹介します。このプロトコルは、血液代謝物の測定のため役に立つかもしれません、中に内分泌マーカー運動。

肘静脈カテーテル法による高強度運動時の血中乳酸および血漿インスリンの評価

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

evaluation-blood-lactate-plasma-insulin-during-high-intensity

Research

JoVE Journal

Immunology and Infection

6.4K ビュー.  Kyoto University. 肘静脈から高強度のインターバル トレーニング中にいくつかの血液サンプルを取得するためのプロトコルをご紹介します。このプロトコルは、血液代謝物の測定のため役に立つかもしれません、中に内分泌マーカー運動。

ビデオ: 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.

補体の枯渇により特定の細胞集団の枯渇

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

免疫・感染学

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.

蛍光活性化細胞選別（FACS）によって特定の細胞集団の精製

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.

運動誘発性リンパ球のアポトーシスの決定のための指先穿刺の採血方法

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トラック運動アッセイによる細胞遊走の定量的評価

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.

抗原は、コンパクトな多分岐ポリグリセロールのグラフト膜によって機能赤血球を保護

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.

過ヨウ素酸シッフ染色で末梢血単核細胞中のグリコーゲンの検出

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.

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

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.

血液処理のための迅速な方法は、ヒト血液中の血漿ペプチドレベルの収量を増やすには

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.

蛍光相関分光法により測定初代リンパ球のプラズマ膜における分子拡散

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.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

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