Os autores gostariam de reconhecer Zack Shue, Casey John e Lynn Cialdella-Kam para a sua assistência durante a otimização deste processo.

<ol>
	<li>Nieman, D. C., 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=Influence+of+pistachios+on+performance+and+exercise-induced+inflammation,+oxidative+stress,+immune+dysfunction,+and+metabolite+shifts+in+cyclists:+a+randomized,+crossover+trial.">Influence of pistachios on performance and exercise-induced inflammation, oxidative stress, immune dysfunction, and metabolite shifts in cyclists: a randomized, crossover trial.</a> PLoS One. 9, e113725 (2014).</li><li>Knab, A. M., 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=Effects+of+a+freeze-dried+juice+blend+powder+on+exercise-induced+inflammation,+oxidative+stress,+and+immune+function+in+cyclists.">Effects of a freeze-dried juice blend powder on exercise-induced inflammation, oxidative stress, and immune function in cyclists.</a> Appl Physiol Nutr Metab. 39, 381-385 (2014).</li><li>Konrad, M., 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=The+acute+effect+of+ingesting+a+quercetin-based+supplement+on+exercise-induced+inflammation+and+immune+changes+in+runners.">The acute effect of ingesting a quercetin-based supplement on exercise-induced inflammation and immune changes in runners.</a> Int J Sport Nutr Exerc Metab. 21, 338-346 (2011).</li><li>Nieman, D. C., 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=Immune+and+inflammation+responses+to+a+3-day+period+of+intensified+running+versus+cycling.">Immune and inflammation responses to a 3-day period of intensified running versus cycling.</a> Brain Behav Immun. 39, 180-185 (2014).</li><li>Nieman, D. C., 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=Carbohydrate+supplementation+affects+blood+granulocyte+and+monocyte+trafficking+but+not+function+after+2.5+h+of+running.">Carbohydrate supplementation affects blood granulocyte and monocyte trafficking but not function after 2.5 h of running.</a> Am J Clin Nutr. 66, 153-159 (1997).</li><li>Tidball, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cell+response+to+acute+muscle+injury.">Inflammatory cell response to acute muscle injury.</a> Med Sci Sports Exerc. 27, 1022-1032 (1995).</li><li>McFarlin, B. K., Williams, R. R., Venable, A. S., Dwyer, K. C., Haviland, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+cytometry+reveals+three+distinct+subsets+of+activated+granulocytes+based+on+phagocytosis+and+oxidative+burst.">Image-based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst.</a> Cytometry A. 83, 745-751 (2013).</li><li>Liao, Y., Liu, P., Guo, F., Zhang, Z. Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+burst+of+circulating+neutrophils+following+traumatic+brain+injury+in+human.">Oxidative burst of circulating neutrophils following traumatic brain injury in human.</a> PLoS One. 8, e68963 (2013).</li><li>McFarlin, B. K., Venable, A. S., Prado, E. A., Henning, A. L., Williams, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+flow+cytometry+technique+to+evaluate+changes+in+granulocyte+function+in+vitro.">Image-based flow cytometry technique to evaluate changes in granulocyte function in vitro.</a> J Vis Exp. , (2014).</li><li>Nieman, D. C., 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=Immune+response+to+obesity+and+moderate+weight+loss.">Immune response to obesity and moderate weight loss.</a> Int J Obes Relat Metab Disord. 20, 353-360 (1996).</li><li>Nieman, D. C., 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=Immune+function+in+female+elite+rowers+and+non-athletes.">Immune function in female elite rowers and non-athletes.</a> Br J Sports Med. 34, 181-187 (2000).</li><li>Ichii, H., 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=Iron+sucrose+impairs+phagocytic+function+and+promotes+apoptosis+in+polymorphonuclear+leukocytes.">Iron sucrose impairs phagocytic function and promotes apoptosis in polymorphonuclear leukocytes.</a> Am J Nephrol. 36, 50-57 (2012).</li><li>Gomes, A., Fernandes, E., Lima, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probes+used+for+detection+of+reactive+oxygen+species.">Fluorescence probes used for detection of reactive oxygen species.</a> J Biochem Biophys Methods. 65, 45-80 (2005).</li><li>Gruger, T., 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=Negative+impact+of+linezolid+on+human+neutrophil+functions+in+vitro.">Negative impact of linezolid on human neutrophil functions in vitro.</a> Chemotherapy. 58, 206-211 (2012).</li><li>Dementhon, K., El-Kirat-Chatel, S., Noel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+in+vitro+model+for+the+multi-parametric+quantification+of+the+cellular+interactions+between+Candida+yeasts+and+phagocytes.">Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes.</a> PLoS One. 7, e32621 (2012).</li></ol>

The authors have nothing to disclose.

Este manuscrito fornece um protocolo passo-a-passo para a determinação dos dois indicadores de granulócitos e monócitos. Foram identificadas algumas etapas que são críticos para o sucesso deste ensaio. Um tal passo é a incubação em banho de gelo que ocorre imediatamente antes da adição de bactérias. arrefecimento exaustiva de todas as amostras irá minimizar o efeito da temperatura sobre a actividade de fagocitose e OB. Um outro passo crucial é a adição de bactérias para cada amostra. A 8: 1 bactérias: ...

A explosão de granulócitos e monócitos fagocitose / oxidativo (OB) ensaio da atividade é uma técnica simples e direta que é frequentemente usado para reunir informações sobre a função imunológica inata após o exercício prolongado e intenso. 1-4 Ao estudar a resposta do sistema imunológico a um estímulo, granulócitos e monócitos são de particular interesse porque eles desempenham um papel central na defesa do hospedeiro e são as primeiras células do sistema imunológico a se acumular no local de infecção. 5 neutrófilos, um tipo de granulócitos, são as primeiras células de se translocar para o músculo danificado tecidos após o exercício intenso. 6 fagocitose ea atividade de OB são as medidas mais comuns da função de granulócitos e monócitos, 7 e são preferidos como indicadores da função celular imune inato por causa de seu papel central, tanto no processo de infecção e o processo de reparação. O ensaio descrito no presente Utili manuscritozes um citómetro de fluxo de base para proporcionar uma determinação quantitativa da fagocitose de granulócitos e monócitos e atividade de OB no sangue total. O uso de sangue inteiro nesta técnica é vantajosa porque permite o ensaio a ser conduzido sem um procedimento de purificação que consomem tempo. Este ensaio pode ser facilmente modificado para acomodar uma variedade de modelos de investigação e de capacidades de laboratório. Por exemplo, Liao et al., Utilizou uma técnica semelhante para demonstrar que a actividade dos neutrófilos OB é aumentada e neutrófilos fagocitose é diminuída após lesão cerebral traumática. 8 Num outro exemplo, McFarlin et al. Descrevem uma modificação elegante desta técnica que utiliza aloficocianina (APC ) -conjugated CD66b e de alto desempenho conjunto rotulagem CD45 APC conjugada com a citometria de fluxo baseada em imagem para classificar os granulócitos em termos de suas atividades fagocitose e OB. 7,9 Nosso grupo de pesquisa tem usado várias adaptações desta technique como um indicador da função imune inata em populações com sobrepeso, obesas, e atléticas por quase 20 anos. 10,11 O texto abaixo irá fornecer o leitor com instruções passo-a-passo para a realização deste ensaio e destacar as áreas que o leitor possa se ​​adaptar para atender às necessidades de suas pesquisas.

<table border="0" cellpadding="0" cellspacing="0" width="1070">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">12 x 75 mm tubes, with cap</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">20170-579</td>
			<td style="width:425px;">You will need 2 tubes per sample (&#34;H&#34; and &#34;F&#34;) plus 3 tubes per batch (for assay controls; &#34;F&#34;, &#34;H&#34;, and &#34;Blood only&#34;).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PBS (10X), pH 7.4</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">70011-044</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bottle top 0.2 &#181;m cellulose acetate filter (500 ml capacity)</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:215px;">09-741-07&#160;</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glucose, powder</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">15023-021</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Citric acid (anhydrous, cell culture tested, plant cell culture tested)</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:215px;">C2404-100G</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Sodium citrate tribasic dihydrate</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:215px;">S4641-25G</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dihydroethidium (Hydroethidine) (HE)</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">D11347</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dimethyl sulfoxide (DMSO) Anhydrous</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">D12345</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">Staphylococcus aureus&#160;(Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">S2851</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Staphylococcus aureus&#160;(Wood strain without protein A) BioParticles, unlabeled</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">S-2859</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">15250-061</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">4.0 mL vacutainer containing 7.2 mg K2EDTA, spray-dried</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">BD367861</td>
			<td style="width:425px;">You will need 1 K2 EDTA blood collection tube per sample.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">4.0 mL vacutainer containing 68 USP units Lithium Heparin, spray-coated</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">BD367884</td>
			<td style="width:425px;">You will need 1 Lithium Heparin blood collection tube per sample.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff CP</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">6605705</td>
			<td style="width:425px;">i.e., hematology analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff Rinse</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547167</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff Fix</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547171</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff WBC Lyse</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547170</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff Hgb Lyse</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547168</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">COULTER Ac&#183;T 5diff Cal Calibrator</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">7547175</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">COULTER Ac&#183;T 5diff Control Plus</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">7547198</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">AcT 5diff Diluent</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547169</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">200 &#181;L extended-length pipette tips</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">37001-526</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Insulated ice pan</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">89198-980</td>
			<td style="width:425px;">For ice water bath.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Open metal tube rack</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">60916-702</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fetal Bovine Serum</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">26140-111</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">TQ-Prep Workstation</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">6605429</td>
			<td style="width:425px;">i.e., automated cell lyse preparation workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">ImmunoPrep Reagent System</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">7546999</td>
			<td style="width:425px;">i.e., RBC lyse/WBC fix reagent system</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Cytomics FC500 MCL Flow Cytometry System with CXP Software</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">626553</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">IsoFlow Sheath Fluid</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547008</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER CLENZ</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8546929</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Flow-Check Fluorospheres&#160;</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">6605359</td>
			<td style="width:425px;">i.e., alignment and fluidics verification fluorospheres</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FlowJo Software</td>
			<td style="width:215px;">FlowJo</td>
			<td style="width:215px;">FlowJo</td>
			<td style="width:425px;">i.e., flow cytometry analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Excel Software</td>
			<td style="width:215px;">Microsoft</td>
			<td style="width:215px;">Microsoft Excel</td>
			<td style="width:425px;">i.e., electronic spreadsheet program</td>
		</tr>
	</tbody>
</table>

measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

o exercício prolongado e intenso tem profundos efeitos sobre a função imunológica inata, incluindo a função natural killer células, resposta mediada por citocinas por macrófagos à infecção viral, e de granulócitos e fagocitose de monócitos e actividade OB. Vários estudos indicam que granulócitos e monócitos fagocitose aumenta significativamente pós-exercício, refletindo a inflamação induzida por lesão muscular e demandas metabólicas. Em contraste, a actividade de gr...

Watch this Scientific Journal Video about Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood at JoVE.com

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

Medição de granulócitos e monócitos fagocitose e oxidativo Atividade Burst no Sangue

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

measuring-granulocyte-monocyte-phagocytosis-oxidative-burst-activity

Research

JoVE Journal

Immunology and Infection

14.0K visualizações.  Appalachian State University. The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

Vídeo: Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

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

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

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

Ativação e Mensuração de NLRP3 inflammasome Atividade Usando IL-1β em células dendríticas humanas Monocyte derivados

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

Imunologia e Infecção

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergética e os oxidativo estourou: Protocolos para o Isolamento e Avaliação de leucócitos humanos e plaquetas

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

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

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

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

Cultura de macrófagos macrófagos Colony estimulante fator diferenciado Humano monócitos derivados

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

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Citometria de fluxo e assassinas naturais celular Lytic Actividade em sangue total humano

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Geração de células dendríticas derivadas de monócitos humanos a partir de sangue inteiro

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Uso de um monócito monocamada Ensaio para Avaliar a fagocitose mediada pelo receptor Fcy

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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 aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

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

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Quantificar o humano Monocyte Chemotaxis In Vitro e linfócitos murino tráfico Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Medição de deformabilidade e heterogeneidade de glóbulos vermelhos no sangue por Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterização de subconjuntos de monócitos humanos por sangue total Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...