Biz (APP605524, 490.037 ve 1.047.082 hibe) Ulusal Sağlık ve Tıbbi Araştırma Konseyi destek fon yny, Avustralya Araştırma Konseyi (DP12010061 hibe), Yenilik Bölümü&#39;nden Avustralya Ulusal İşbirlikçi Araştırma Altyapısı Strateji ve Eğitim yatırım fonu, Sanayi Bilim ve Araştırma. PML Avustralya Lisansüstü ödül bir alıcı olduğunu.

Tüm işlemler Macquarie Üniversitesi politikaları doğrultusunda yürütülen ve Ulusal Sağlık ve Tıbbi Araştırma Konseyi (NHMRC) uygulama Avustralya kod uyduğu bulundu. iş yok ARA 2012/017 onaylı ve Macquarie Üniversitesi Hayvan Etik Komitesi&#39;nden alınan anlaşma Etik altında yapıldı. Aksi belirtilmedikçe tüm deneyler SJL / J farelerinde gerçekleştirilmiştir. 1. Fare ve Deneysel sıtma enfeksiyonu <ol><li> 12:12 saat ışık-karanlık çevrimi ile kontrol edilen bir sıcaklıkta (21 ° C) altında Ev fareler. </li><li> Kriyoprezerve P. bir kısım çözülme % 5 parazite eritrosit (prbcS) ve C57BL / 6 donör farenin periton boşluğuna 200 ul enjekte ile chabaudi adami DS. </li><li> Bu protokolün 4 - Bölüm 3 açıklandığı gibi flow sitometri kullanılarak 2 gün - her 1 donör fare paraziteminin izleyin. Donörler 5 ulaştığında -% 15 oranında paraziteminin aşağıdaki gibi, kalp ponksiyonu ile kan toplamak: <ol><li>Aspire heparin çözeltisi ile (fare Ringer çözeltisi içinde, 300 U / ml heparin (MTR) (154 mM NaCI, 5.6 mM KCI, 1 mM MgCl2, 2.2 mM CaCl2, 20 mM HEPES, 100 ul 10 mM glukoz, 30 U / ml Heparin, pH 7.4, 0.22 uM filtre 26 G iğne ile bir 1 ml şırınga içine)) sterilize edilmiştir. </li><li> Pedal çekilme tepkisi ve kornea refleksleri yokluğu ile anestezi istenen derinliği teyit% 5 isofluorane ile inhalasyon yoluyla fare anestezisi. </li><li> Kaburga ile, sadece dirsek altında yan fare ve dikey uç iğne yerleştirin ve kalbine. Yavaş yavaş şırınga pistonu çekin ve 0,5 kadar iğne döndürmek - 1 ml kan elde edilir. </li><li> Anestezi altında servikal dislokasyon ile insani ötenazi yapın. </li></ol></li><li> Kurban ne zaman donör% 10 parazitemya oldu, eğer kan 9 x 10 5 içerecektir yani 9 x 10 6 RBC / ul (bir kan sayımı varsayarak donör kan hacmi başına prbcS hesaplayınpRBC / ul). Krebs parazitik kan seyreltin (25 mM NaHCO 3, 1.2 mM NaH 2 PO 4, 1.2 mM MgCl2, 2.5 mM CaCl2,% 0.2 glukoz, pH 7.2, steril 0.22 mikron filtre 126 mM NaCI, 2.5 mM KCI) tamponlu tuz konsantrasyon 200 ul başına 1 x 10 4 prbcS ve RBC etiketleme ve transfüzyon deney için gerekli olan farelerin periton boşluğuna 200 ul enjekte böylece. </li><li> Bu protokolün 4 - Bölüm 3 açıklandığı gibi flow sitometri kullanılarak 2 gün - her 1 paraziteminin izleyin. </li></ol> Eritrosit ve Transfüzyon 2. Etiketleme <ol><li> Aşama 1.3 &#39;de tarif edildiği gibi kardiyak delme ile heparinize edilmiş farelerden kan toplamak ve buz üzerine yerleştirin. Her zaman, 4 ° C&#39;de kan tutun. Fare başına yaklaşık 200 ul kan toplayın enjekte edilecek. Gerekirse iki numune içine kan bölünmüş ve arzu edilen bir örnek ele. Not: tedavi SAMP Bu şekilde, istilale bir kontrole kıyasla olabilir, işlenmemiş numune (şematik için Şekil 1). </li><li> Her floresan RBC etiketinin 2x çözeltisi hazırlayın. <ol><li> Konsantrasyon 20 ug / ml olacak şekilde MTR içinde Atto 633-N-hidroksisüksinimid (Atto 633-NHS) içindeki bir 2 mg / ml stok çözeltisi ile seyreltilir. </li><li> Konsantrasyonu 250 ug / ml olacak şekilde MTR Biotin-N-hidroksisüksinimid (Biotin-NHS), bir 25 mg / ml stok çözeltisi ile seyreltilir. </li></ol></li><li> Iki tüp içine kan örnekleri bölme ve bir boru ve başka boru 2x Biotin-NHS, eşit hacimde 2x Atto 633-NHS çözeltisi eşit hacimde ekleyin. Hemen karıştırın. Sabit yavaş bir karıştırma ile 1 saat süreyle 4 ° C&#39;de inkübe edin kan, ya da seçenek olarak, her 10 örnek karışımı - 15 dakika. </li><li> Aşağıdaki gibi MTRC (MTR +% 0.5 sığır serum albümini (BSA), steril 0.22 mikron filtre) kan etiketli 3 kez yıkayın: <ol><li> 5 dakika boyunca 750 x g&#39;de MTRC en az 2 hacim, santrifüj ekleme süpernatant kaldırmak ve resuspenMTRC en az 2 hacim pelet d. 2 kez daha tekrarlayın. </li><li> 5 dakika ve 750 xg&#39;de son yıkama, santrifüj sonrasında farklı kombinasyonlarda topak haline kan birleştirmek (yani, işlenmemiş Atto 633 / tedavi Biotin, işlenmemiş Biyotin / Atto 633 tedavi). Enjekte edilecek, fare başına 200 ul bir hacme kadar telafi etmek MTR ekleyin. </li></ol></li><li> Aşağıdaki gibi intravasküler kemirgen sıtma enfekte edilen farelerin ya da enfekte olmamış kontrollere içindeki işaretli kan enjekte: <ol><li> Parazit schizogony zirvesinde% 15 parazitemya - 2 de transfüzyonu gerçekleştirin. NOT: Bu karanlık döngüsü boyunca yaklaşık yarım meydana gelir (yani, 12 arasında - ters ışık döngüsü 14:00 - normal ışık döngüsü 02:00, veya 12). </li><li> 5-10 dakika için bir ısı lambası kullanarak Sıcak fareler, kan akışını artırmak, ancak fareler yanmamasına tedbirleri almak. Frenleyici bir cihaz fareler yerleştirin ve intravasküler 200 ul etiketli kan enjekte (yaklaşık 1-2 x 10 9 RBKuyruk damarından Cs). </li></ol></li><li> Bölüm 3&#39;te tarif edildiği gibi enjeksiyondan sonra farklı zaman noktalarında kan örnekleri toplamak. Not: istila oranları alıcı farenin parazitemi bağlı olarak, enjeksiyondan sonra en az 10 dakika boyunca ölçülebilir. </li></ol> Akış Cytometry Kan Örneklerinin ve Hazırlık 3. Koleksiyonu <ol><li> Aşağıdaki gibi deney gününde, boyama numune başına çözeltisi artı ekstra 50 ul hazırlanması: <ol><li> Dimetil sülfoksit içinde, -20 ° C (DMSO) saklanan 6 mM JC-1 bir kısım defrost. </li><li> 37 ° C sıcak bir 50 numune başına MTRC ul, artı ilave. </li><li> Nihai konsantrasyon 12 uM olacak şekilde sürekli olarak önceden ısıtılmış MTRC vorteks iken, JC-1 ekleyin. Bu JC-1 agrega oluşumunu azaltmak için yapılır; agrega formu yoksa, bu boyama önlemek gibi değil santrifüj tüp yapmak. NOT: toplama maddesinin küçük bir miktarı sonuçlarını etkilemez. </li><li> Anti EkleCD45 APC eFluor 780 ve anti-CD71 PerCP eFluor 710 nihai konsantrasyon 1 ug / ml olacak şekilde. Etiketli eritrosit Deney durumunda nihai konsantrasyon 1 ug / ml olacak şekilde streptavidin PE-Cy7 ekleyin. </li></ol></li><li> Kuyruk içindeki kan damarı kuyruk veya laserasyon ucu amputasyon ile kuyruk kanama gerçekleştirin. Küçük tartmak tekne veya cam slayt üzerine kuyruk kan bir damla yerleştirin. Kan toplama sonra, kanama durduktan emin olun. Boyama çözeltisi 50 ul halinde kuyruk kan Pipet 3 ul 37 ° C&#39;ye önceden ısıtılmış ve 20 dakika boyunca 37 ° C&#39;de numune inkübe edin. </li><li> (355 nm lazer kullanılıyorsa) damıtılmış su içinde -20 ° C&#39;de saklandı, 4 mM Hoechst 33342 ya da (405 nm lazer kullanılıyorsa) damıtılmış su içinde -20 ° C&#39;de saklandı, 2 mM Hoechst 34580 bir kısım defrost. Sırasıyla MTRC içinde Hoechst bir 4 uM ya da 2 uM çözeltisi, örnek başına 500 ul hazırlanması, ayrıca ilave. </li><li> 4 mcM Hoechst 500 ul ekleyin33342 veya numune 2 uM, Hoechst 34580 ve 20 dakika boyunca oda sıcaklığında inkübe edilir. </li><li> 4 ° C&#39;de 3 dakika boyunca 750 xg, ıskarta süpernatant, Santrifüj hücreleri 700 ul MTRC ekleyin ve flow sitometri ile analiz. </li></ol> 4. Akım Sitometri <ol><li> Bir 355/488/633 nm lazer veya 405/488/633 nm lazer cihazı kullanarak örnekleri analiz edin. </li><li> Analiz hemen önce 35 mcM hücre süzgecinden Filtre örnekleri. Örnekler ve yeniden kullanım arasında damıtılmış su ile hücre süzgecinden durulayın. </li><li> (- Numune başına 10,000,000 toplam olaylar genellikle 1.000.000) küçük nüfusu en az 500 olayları içerecek şekilde yeterli olayları kaydedin. </li><li> 355 nm lazer kullanarak Hoechs 33.342 Excite ve bir 460/50 filtresi aracılığıyla olayları algılar. 405 nm lazer kullanarak Hoechs 34.580 Excite ve bir 460/50 filtresi aracılığıyla olayları algılar. Excite JC-1 anti-CD71 PerCP eFluor 710 ve Streptavidin PE-Cy7 488 nm lazer kullanarak ve 530/40 ile olayları tespit, 692/40 ve 750LP ilgili filtrely. Bir 633 nm lazer kullanarak ATTO 633 ve anti-CD45 APC eFluor 780 Excite ve sırasıyla 670/30 veya 750LP filtresi aracılığıyla olayları algılar. </li><li> Aşağıdaki gibi analiz yazılımı sitometri uygun akışını kullanarak analiz ve tazminat gerçekleştirin: <ol><li> Bütün hücreleri seçin ve FSC / SSC özellikleri (Şekil 2A) dayalı gürültü, enkaz ve trombosit dahil değildir. Her iki tetik darbe genişliği (Şekil 2B) veya yükseklik oranı (Şekil 2C) FSC pik alanı kullanılarak dayalı tek hücreleri seçin. </li><li> Olgun eritrositlerde seçin ve PerCP eFluor 710 ve APC eFluor 780 kanalları (Şekil 2B) negatif floresan dayalı RBC atalarıdır ve lökositler, dışarıda. </li><li> Etiketli RBC deneyi gerçekleştirmek ise, PE-Cy7 ve ATTO 633 floresan dayalı etiketli eritrosit her nüfusu seçin ve bu ayrı (Şekil 3A) analiz. </li><li> (Ikisi de JC-1 ve Hoechst pozitif floresan dayalı prbcS seçin <strOng&gt; Şekil 2E-H) &#39;. </li></ol></li></ol> 5. Hesaplamalar ve İstatistik <ol><li> Eritrosit toplam sayısına göre prbcS sayısına bölünmesiyle paraziteminin hesaplayın (yani, kapı G4 olayların sayısına bölünmesiyle kapı Q2 olayların sayısı). İki etiketli nüfusun işgali tahlil paraziteminin yapılırken (bu popülasyonda etiketli eritrosit sayısına göre etiketli prbcS sayısına bölünmesiyle hesaplanır, yani kapıları L1 ve Q2 hem de olan olayların sayısı, olayların sayısına bölünmesiyle kapı L1). NOT: Etiketli parazitemisi ölçüde böyle endojen parazitemya ve dolaşımdaki merozoit sayısı gibi faktörlere bağlı olarak değişir. Bu nedenle, her bir fare kontrol etiketli nüfusun parazitemi bölünmesiyle tedavi etiketli nüfusun parazitemi olarak hesaplanır parazitemi oranını rapor yararlıdır. Olası boya efektleri düzeltmek için, rapor sonuçları birİki boya kombinasyonları arasında ortalama parazitemisi oranı s. </li><li> Varsayımsal ortalama olarak 1 ile bir örnek t-testi kullanılarak oranlarının istatistiksel olarak anlamlı belirleyin. </li></ol>

<ol>
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A., 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=RBC+barcoding+allows+for+the+study+of+erythrocyte+population+dynamics+and+P.+falciparum+merozoite+invasion.">RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.</a> PLoS One. 9 (7), e101041 (2014).</li><li>Malleret, B., 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=A+rapid+and+robust+tri-color+flow+cytometry+assay+for+monitoring+malaria+parasite+development.">A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.</a> Sci Rep. 1 (118), (2011).</li><li>Jimenez-Diaz, M. B., 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=Quantitative+measurement+of+Plasmodium-infected+erythrocytes+in+murine+models+of+malaria+by+flow+cytometry+using+bidimensional+assessment+of+SYTO-16+fluorescence.">Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.</a> Cytometry A. 75 (3), 225-235 (2009).</li><li>Jimenez-Diaz, M. B., 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=Improvement+of+detection+specificity+of+Plasmodium-infected+murine+erythrocytes+by+flow+cytometry+using+autofluorescence+and+YOYO-1.">Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.</a> Cytometry A. 67 (1), 27-36 (2005).</li><li>Jun, G., Lee, J. S., Jung, Y. J., Park, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+Plasmodium+parasitemia+by+flow+cytometry+and+microscopy.">Quantitative determination of Plasmodium parasitemia by flow cytometry and microscopy.</a> J Korean Med Sci. 27 (10), 1137-1142 (2012).</li><li>Lelliott, P. M., Lampkin, S., McMorran, B. J., Foote, S. J., Burgio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometric+assay+to+quantify+invasion+of+red+blood+cells+by+rodent+Plasmodium+parasites+in+vivo.">A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.</a> Malar J. 13 (1), 100 (2014).</li><li>Morohashi, K., 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=Structural+and+functional+abnormalities+in+the+spleen+of+an+mFtz-F1+gene-disrupted+mouse.">Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.</a> Blood. 93 (5), 1586-1594 (1999).</li><li>Shet, A. S., 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=Morphological+and+functional+platelet+abnormalities+in+Berkeley+sickle+cell+mice.">Morphological and functional platelet abnormalities in Berkeley sickle cell mice.</a> Blood Cells Mol Dis. 41 (1), 109-118 (2008).</li><li>Duraisingh, M. 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=Phenotypic+variation+of+Plasmodium+falciparum+merozoite+proteins+directs+receptor+targeting+for+invasion+of+human+erythrocytes.">Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes.</a> EMBO J. 22 (5), 1047-1057 (2003).</li><li>Okoyeh, J. N., Pillai, C. R., Chitnis, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+field+isolates+commonly+use+erythrocyte+invasion+pathways+that+are+independent+of+sialic+acid+residues+of+glycophorin+A.">Plasmodium falciparum field isolates commonly use erythrocyte invasion pathways that are independent of sialic acid residues of glycophorin A.</a> Infect Immun. 67 (11), 5784-5791 (1999).</li><li>Dolan, S. A., Miller, L. H., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+switching+mechanism+in+the+invasion+of+erythrocytes+by+Plasmodium+falciparum.">Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.</a> J Clin Invest. 86 (2), 618-624 (1990).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bu, in vivo örneklerin her ikisi de parazitemi ve Merozoit istilası ölçümü için bir yöntem tanımlanmıştır. Parazitemi ölçümü açısından, bu yöntem, ve böylece yanlış pozitif olayların sayısını azaltarak, prbcS ayırt edilebilir HJ-eritrositlerde önceki yöntemlerin 10-13 üzerinde bir avantaj sağlar. HJ-eritrosit insanlarda genellikle nadir olmakla birlikte, bazı çalışmalar kemirgen parazitemya doğru ölçümü için önemli olan bu hücreler ve prbcS arasındaki ayr?...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>JC-1 Dye</td>
			<td>Life Technologies</td>
			<td>T-3168</td>
			<td>Store small aliquots of 6mM stock solution at -20 &#176;C in DMSO</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 APC-eFluor 780</td>
			<td>eBioscience</td>
			<td>47-0451-80</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD71 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0711-80</td>
			<td>Clone R17217</td>
		</tr>
		<tr>
			<td>Atto 633 NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>1464</td>
			<td>Atto 633-NHS. Store a 2mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>21335</td>
			<td>Biotin-NHS. Store a 25mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>Streptavidin PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4317-82</td>
			<td>Streptavidin PE-Cy7</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H478</td>
			<td></td>
		</tr>
		<tr>
			<td>35&#181;M filter cap tubes</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD LSRFortessa</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD FACSAria II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD Influx</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: CyAn ADP Analyzer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Parazitemya ölçümü. Parazitemya ölçümü için kan hücreleri ilk seçilmelidir ve gürültü, enkaz ve trombositler FSC / SSC özellikleri (Şekil 2A) dayalı hariç. Kullanılan sitometresinde bağlı olarak, tek hücreler, daha sonra alan oranı (Şekil 2C) için iki pals genişliği (Şekil 2B), veya FSC pik yüksekliğine göre seçilmelidir. Etkinlik lökositlerin oluşmalıdır kalan APC eFluor 780 için pozitif boyandı, PerCP eFl...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

kırmızı kan hücrelerinin içinde sıtma paraziti girdiği, ve çoğaltır. Merozoit işgali ve parazitemya doğru değerlendirme sıtma enfeksiyonu seyrini değerlendirmede nedenle çok önemlidir. Burada sıtma bir fare modelinde bu parametrelerin ölçülmesi için tabanlı protokol sitometri akışı tarif eder.

Akış Cytometry tarafından Kemirgen Plasmodium parazitemi Vivo Değerlendirilmesi ve Merozoit Invasion

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.5K Görüntüleme.  Macquarie University.  kırmızı kan hücrelerinin içinde sıtma paraziti girdiği, ve çoğaltır. Merozoit işgali ve parazitemya doğru değerlendirme sıtma enfeksiyonu seyrini değerlendirmede nedenle çok önemlidir. Burada sıtma bir fare modelinde bu parametrelerin ölçülmesi için tabanlı protokol sitometri akışı tarif eder. 

Video: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Sabit ve Yaşam Hücreler Sitometrisi ile Calpain Aktivitesi Ölçme

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

İmmünoloji ve Enfeksiyon

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Akış Sitometri Yaralı Omurilik Doku Bağışıklık Hücreleri Kantitatif Değerlendirilmesi: Hücre Arıtma Yöntemi Roman kullanın

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Insan In Vitro Bağışıklık Dengesizlik Erken Devlet Tanıma Tarama Aracı Olarak Bastırılması

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Akım Sitometri tarafından Timik Pozitif ve Negatif Seçim incelenmesi

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Akış Sitometrisi ile Sıçan Böbrek gelen Makrofagların Fenotipik Karakterizasyonu

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi<em&gt; İn Vivo</em&gt; A Akış Sitometri bazlı analiz kullamlarak

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10 parçacık bağlı Bet v 1 Allergen Akışı Sitometrik Analizi

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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).

İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

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

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

İki-fluorochrome tarafından yedi bağışıklık hücre alt kümeleri ayrımcılık Akış Sitometresi

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...