Die Autoren möchten Dr. P. Karagiannis für die Zeitung lesen und Professor T. Hoyo für seine technische Unterstützung zu erkennen.

Alle hier beschriebene Methoden wurden von der lokalen Ethikkommission genehmigt (Doshisha Universität Ethikkommission: 15033), und waren in strikter Übereinstimmung mit den Standards der Deklaration von Helsinki. Informierte Zustimmung wurde von allen Teilnehmern erhalten. Teilnehmer waren männliche Freizeitsportler (Alter, 20.1 ± 1,2 Jahre; Gewicht, 69,7 ± 6,2 kg; Höhe, 176.1 ± 5,8 cm; Körper Fett 13,6 ± 1,5 % und VO2max, 52.6 ± 6,6 mL/min1schnitzelfleisch1).
1. Aufbau des Protokolls Übung
Hinweis: Die vorliegende Studie bestand aus der Bewertung von Blut hormonelle und Metaboliten Dynamik durch Blutentnahme aus der Vene Antecubital während ein HIIE Protokoll.
<ol><li>Fragen Sie das Thema, eine warme, mit 5 min des Radsports in der Ergometer bei niedriger Intensität durchzuführen. Lassen Sie nach dem Warm-up das Thema für 5 min ruhen.</li>
	<li>Bitte das Thema der folgenden HIIE in das Bein Fahrradergometer durchzuführen: drei Anfälle von 30 s hoch-intensives Training, getrennt durch 4 min. Rest (Abbildung 1). Hinweis: Die 30 s-Kämpfe bei gleicher Intensität zu 90 % den macht-Durchschnitt von einer zuvor durchgeführten Wingate-Test festzulegen. Finden Sie an anderer Stelle8 für eine vollständige Beschreibung der Wingate-Test.</li>
	<li>Um die Arbeitsbelastung für die 30 s-Kämpfe zu berechnen, verwenden Sie die folgenden Gleichungen: Arbeit = Kraft x Abstand (Gleichung 1) Kraft = Masse x Beschleunigung (Gleichung 2) Arbeit = (Masse x Beschleunigung) x Abstand (Gleichung 3) Hinweis: In diesem Modell ist die Masse der Arbeitslast (kg), und die Beschleunigung beträgt 9,81 m/s2. Die Entfernung beträgt der Durchmesser der Räder (1,622 m). Da das Rad 3,7 zeitgesteuerte /peal Drehung dreht, ist der Abstand pro Pedal Umdrehung 3,7 x 1.662. Andererseits, weil die Trittfrequenz 90 u/min-1ist, haben wir ein Pedalabstand mit 90 zu multiplizieren. Arbeit = Arbeitslast x 9,81 x 3,7 x 1.622 x 90 (Gleichung 4) Belastung = Arbeit / (9,81 x 3,7 x 1.622 x 90) (Gleichung 5)</li>
</ol>(2) Katheterisierung
Hinweis: Rohre und vacutainer sollten im Vorfeld zu vermeiden, dass Sie Zeit verlieren beschriftet werden.
<ol><li>Wickeln Sie einen Druckverband am Arm für die Katheterisierung ausgewählt. Um dies zu tun, legen Sie eine elastisches Band auf der Rückseite der gewählten Arm. Dehnen die Band, machen Sie eine Schleife mit der Band um den Arm und binden Sie die elastischen.</li>
	<li>Wählen Sie die sichtbaren Antecubital Vene und Sterilisieren Sie die Haut mit Alkohol oder Ethanol.</li>
	<li>Führen Sie die Spitze der sterilisierte Kanüle in die Vene in einem ungefähren Winkel von 15° mit der Haut. Hinweis: Die Kanüle sollte von der proximalen, distalen Richtung eingefügt werden. Wenn Kanülierung erfolgreich ist, sollte eine retrograde Entnahme von Blut beobachtet werden.</li>
	<li>Sichern Sie die Kanüle mit der Hand, und entfernen Sie die Nadel sanft. Hinweis: Um den Winkel der Nadel während der Übung zu halten, können Baumwolle Gewebe zwischen Haut und Nadel verwendet werden. Befestigen Sie diese Struktur mit einem Band.</li>
	<li>Verbinden Sie ein Vacutainer mit Heparin und aspirat für die erste Blutprobe (ca. 2,5 mL). Unmittelbar nach kühlen Sie der Blutentnahme indem man es in eine Eisbox für nicht länger als 40 Minuten. Bitten Sie ein Assistent um Hilfe, wenn Blutgaswerte analysiert werden sollen.</li>
	<li>Schließen Sie einen Schlauch an den Katheter und Spritzen Sie Heparin auf die volle Länge des Rohres um eine Blutgerinnung zu vermeiden. Schließen Sie den Schmetterling.</li>
	<li>Mit Klebeband, stabilisieren Sie den Katheter. Stellen Sie sicher, dass an dieser Stelle die Arm-Beweglichkeit des Themas mit der Blutentnahme nicht stört. Um diese Bedingung zu bestätigen, werden Sie sicher, dass die Nadelposition jede Bewegung nicht betroffen ist.</li>
</ol>3. Blut Samplings
<ol><li>Entfernen Sie das Heparin mithilfe einer Vacutainer bis es nur Blut in den Katheter gibt. Wenn Blut Rückblende beobachtet wird, sammeln Sie die Probe wie in Schritt 2.5 beschrieben.</li>
	<li>Spritzen Sie nach der Probenentnahme Heparin wieder in den Katheter Schlauch Blutgerinnung zu vermeiden. Nach dieser Aktion kann Schritt 2.5 für mehrere Proben Sammlung wiederholt werden.</li>
	<li>Nach die letzte Probe erhoben werden, sorgfältig die Kanülierung Rohr zu entfernen und die Haut mit Alkohol reinigen. Um mögliche Blutungen zu vermeiden, bitten Sie, das Thema weiterhin sterilisiert Baumwolle an der Stelle wo die Katheterisierung gemacht wurde.</li>
	<li>Nachdem Blut in die Vacutainer-Röhrchen gesammelt, sorgfältig etwa 1,5 mL Blut zu einem 1,5 mL Schlauch und halten Sie die Rohre auf dem Eis.</li>
	<li>Maßnahme-Blut-Laktat aus dem restlichen Blut mit einem Laktat-Detektor. Am selben Tag erhalten Sie dem Plasma der Blutproben (rund 1,5 mL Blut).</li>
	<li>Für Plasma-Teilung Zentrifugieren der Blutproben für 5 min bei 5000 X g bei 4 oC, und Trennen des Überstands in ein neues 1,5 mL-Tube.</li>
	<li>Wiederholen Sie die Zentrifugation noch einmal um das Plasma zu reinigen.</li>
	<li>Nach der Trennung Plasma speichern Sie die Proben bei-80 oC bis die Hormon-Analyse.</li>
	<li>Das Plasma-Insulin mit einem ELISA-Kit wie pro des Herstellers Protokoll (siehe Tabelle der Materialien) zu messen.</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>

Die Autoren haben nichts preisgeben.

In dieser Studie die Antecubital Vene war katheterisiert und Blutproben wurden erfolgreich gesammelt, während ein hohes Intervall-Trainings. Als ein Beispiel für die Verwendung dieser Methodik wurden Blutproben für Plasma-Insulin für Blut-Laktat und (nach Plasma Trennung) analysiert. Der Hauptvorteil dieser Methode ist, dass es die Sammlung von Blutproben in kurzen Zeiträume ermöglicht. Zur gleichen Zeit hat es einige Einschränkungen. Zum Beispiel die begrenzte Übung Modalitäten, die seine Verwendung; die Anzahl...

Die Messung der endokrinen und metabolischen Marker während der körperlichen Aktivität ist von Bedeutung für das Verständnis der physiologischen Auswirkungen der verschiedenen Übung Modalitäten. Während einige Übung Modalitäten (z. B. hoher Intensität Intervall Übung (HIIE)) schwanken Blut-Metaboliten und Hormonspiegel in relativ kurzer Zeit1,2,3.
Viele Sportarten (z.B. Fußball, Basketball und Rugby) bestehen aus kurzfristig hoch-intensives Training durch niedrig intensivem Training und/oder passive Perioden4,5getrennt sind. HIIE zeigt Ähnlichkeiten mit den oben genannten Sportarten und ist daher weithin als eine Trainingsmethode für Athleten6verwendet. Darüber hinaus wegen seiner Ähnlichkeit mit Sport, wurde HIIE als Modell zur Übung Physiologie7zu studieren. Blutabnahme ist entscheidend für die physiologische Variablen zu messen, und es wird häufig verwendet für Forschungszwecke und die Beurteilung der körperlichen Verfassung. Blutabnahme während HIIE kann jedoch aufgrund der kurzen Zeitspanne zwischen Musterkollektionen technisch schwierig sein. Als Lösung für dieses Problem präsentieren hier wir eine Methode, um Blutproben von venösen Katheterisierung Antecubital kurz erhalten Zeiträume während Themen HIIE durchführen.

<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

Die Messung der endokrinen und metabolischen Marker während der körperlichen Aktivität ist von Bedeutung für das Verständnis der physiologischen Auswirkungen der verschiedenen Übung Modalitäten. Während einige Übung Modalitäten (z. B. hoher Intensität Intervall Übung), Blut Metaboliten und Hormonspiegel ändern in kurzen Zeiträumen. In der vorliegenden Studie beschreiben wir eine Methode, um die Antecubital Vene Katheterisierung ermöglicht die Sammlung von mehreren Blutproben während des Trainings. Insulin und venösen Laktat-Konzentrationen wurden während des intensiven Trainings durch die Anwendung des beschriebenen Verfahrens gemessen. Die Übung bestand aus drei 30 s Anfälle von hoch-intensives Training durch 4 min Erholung getrennt. Nach der letzten Erholungsphase wurde ein Wingate-Test durchgeführt. Blutproben aus der Antecubital Vene wurden vor und nach jedem 30 s-Anfall und vor und nach dem Wingate-Test erhalten. Dadurch war es möglich, die Plasma-Insulin und venöses Blut Laktat Variationen während des Trainings zu bewerten.

Als repräsentative Ergebnisse zu erzielen haben wir dieses Protokoll Blut Laktat und Insulin Variation in sechs Fächern während einer HIIE in einem Fahrradergometer gemessen. Spitzenleistung und Durchschnittsleistung für den Wingate-Test wurden 732 ± 120 und 549 ± 84 Watt, beziehungsweise. Durchschnittsleistung wurde verwendet, um die Ausgangsleistung (494 ± 75 Watt) während die 30 s-Anfälle von den HIIE zu berechnen.
<...

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

Hier präsentieren wir ein Protokoll, um mehrere Blutproben aus der Antecubital Vene während hochintensivem Intervalltraining zu erhalten. Dieses Protokoll kann nützlich sein für die Messung von Blut Metaboliten und endokrine Marker während der Übung.

Auswertung der Blut-Laktat und Plasma-Insulin im hoch-intensives Training von Antecubital Vene Katheterisierung

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 Aufrufe.  Kyoto University. Hier präsentieren wir ein Protokoll, um mehrere Blutproben aus der Antecubital Vene während hochintensivem Intervalltraining zu erhalten. Dieses Protokoll kann nützlich sein für die Messung von Blut Metaboliten und endokrine Marker während der Übung.

Serie: 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.

Depletion spezifischer Zellpopulationen durch Ergänzung Depletion

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

Forschung

Immunologie und Infektion

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.

Reinigung von spezifischen Zellpopulation durch 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 ...

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.

Finger-Stick Blutprobenentnahme Methodik zur Bestimmung der Übung-induzierte Lymphozyten Apoptose

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.

Eine quantitative Auswertung der Zellmigration durch die Phagokinetic Spur Motilität Assay

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.

Antigene geschützten funktionellen Red Blood Cells durch die Membran Pfropfung von Compact hyperverzweigten Polyglycerinen

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.

Erkennen von Glykogen in peripheren mononukleären Blutzellen mit Perjodsäure-Schiff-Färbung

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.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

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.

Eine schnelle Methode zur Blutverarbeitung die Ausbeute an Plasma-Peptidkonzentrationen im menschlichen Blut zu erhöhen

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.

Molekulare Diffusion in Plasmamembranen von primären Lymphozyten mittels Fluoreszenz-Korrelations-Spektroskopie gemessen

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 erhöht die Effizienz der Lentivirale Transduktion von murinen und menschliche primäre NK-Zellen

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