Name: flow cytometry to estimate leukemia stem cells in primary acute myeloid leukemia and in patient-derived-xenografts, at diagnosis and follow up
Uploaded: 2018-03-26T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up at JoVE.com

Bu eser kısmen Fransızca Derneği Laurette Fugain, LigueContre le kanser (Kuzey Merkezi), Fondation de France (lösemi Komitesi), SIRIC Oncolille ve Fransız Ulusal Kanser Enstitüsü tarafından desteklenmiştir.

Avrupa standartlarında bilimsel amaçlar için hayvan kullanımı için takip ediyoruz. Bu çalışmada yerel Etik Komitesi (#3097-2015120414583482v3) tarafından kabul edildi.
1. numune hazırlama
<ol><li>Kemik iliği (2 mL) de novo AML antikoagülan ethylenediaminetetraacetic asit (EDTA) içeren tüpler 1.8 mg-mL kemik iliği bir konsantrasyon, toplamak.</li>
	<li>Ficoll ayırma, alyuvar ve granülosit, mononükleer hücreler olarak izole etmek için yoğunluğu bir degrade renk ...

<ol>
	<li>Shlush, L. I., 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=Tracing+the+origins+of+relapse+in+acute+myeloid+leukaemia+to+stem+cells.">Tracing the origins of relapse in acute myeloid leukaemia to stem cells.</a> Nature. , (2017).</li><li>Terwijn, 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=Leukemic+stem+cell+frequency:+a+strong+biomarker+for+clinical+outcome+in+acute+myeloid+leukemia.">Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.</a> PLoS One. 9 (9), e107587 (2014).</li><li>Jordan, C. 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=The+interleukin-3+receptor+alpha+chain+is+a+unique+marker+for+human+acute+myelogenous+leukemia+stem+cells.">The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.</a> Leukemia. 14 (10), 1777-1784 (2000).</li><li>van Rhenen, 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=The+novel+AML+stem+cell+associated+antigen+CLL-1+aids+in+discrimination+between+normal+and+leukemic+stem+cells.">The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.</a> Blood. 110 (7), 2659-2666 (2007).</li><li>Bonardi, F., 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+proteomics+and+transcriptomics+approach+to+identify+leukemic+stem+cell+(LSC)+markers.">A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.</a> Mol Cell Proteomics. 12 (3), 626-637 (2013).</li><li>Kikushige, Y., Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+TIM-3+as+a+Leukemic+Stem+Cell+Surface+Molecule+in+Primary+Acute+Myeloid+Leukemia.">Identification of TIM-3 as a Leukemic Stem Cell Surface Molecule in Primary Acute Myeloid Leukemia.</a> Oncology. 89, 28-32 (2015).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nat Med. 3 (7), 730-737 (1997).</li><li>Kersten, 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=CD45RA,+a+specific+marker+for+leukaemia+stem+cell+sub-populations+in+acute+myeloid+leukaemia.">CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.</a> Brit J Haematol. 173 (2), 219-235 (2016).</li><li>Goardon, N., 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=Coexistence+of+LMPP-like+and+GMP-like+leukemia+stem+cells+in+acute+myeloid+leukemia.">Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia.</a> Cancer Cell. 19 (1), 138-152 (2011).</li><li>Ng, S. W., 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+17-gene+stemness+score+for+rapid+determination+of+risk+in+acute+leukaemia.">A 17-gene stemness score for rapid determination of risk in acute leukaemia.</a> Nature. 540 (7633), 433-437 (2016).</li><li>Shultz, L. D., 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+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R+gamma+null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.</a> J. Immunol. 174 (10), 6477-6489 (2005).</li><li>Roloff, G. W., Lai, C., Hourigan, C. S., Dillon, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Advances+in+the+Measurement+of+Residual+Disease+in+Acute+Myeloid+Leukemia.">Technical Advances in the Measurement of Residual Disease in Acute Myeloid Leukemia.</a> J Clin Med. 6 (9), (2017).</li><li>Swamydas, S., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Purification+and+Labeling+of+Mouse+Bone+Marrow+Neutrophils+for+Functional+Studies+and+Adoptive+Transfer+Experiments.">Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments.</a> J Vis Exp. (77), e50586 (2013).</li><li>Gabert, J., 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=Standardization+and+quality+control+studies+of+'real-time'+quantitative+reverse+transcriptase+polymerase+chain+reaction+of+fusion+gene+transcripts+for+residual+disease+detection+in+leukemia+-+a+Europe+Against+Cancer+program.">Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.</a> Leukemia. 17 (12), 2318-2357 (2003).</li></ol>

Enstrüman ayarlarını her gün optik uyum, sıvı sistemi, optik duyarlılık ve standardizasyon işlevlerin doğru için doğrulanır membran veya Akış Sitometresi tarafından sitoplazmik işaretleri doğru ve tekrarlanabilir değerlendirilmesi gerektirir kalibrasyon boncuk kullanarak.
CD90 ve CD45RA göre çok parametrik Akış Sitometresi Analizi kullanarak AML blast hücre popülasyonlarının tespiti nispeten basit ve güvenilir bir yöntemdir. Bu analizi kritik bir adımda doğru CD3...

Akut Miyeloid Lösemi (AML) Lösemi ilişkili ölümlerin en sık nedenidir. Başlangıçta, AML farklılaşma, blast hücrelerinin sonraki birikimi tutuklanması tarafından karakterize ve üretim fonksiyonel hematopoetik öğelerinin azaltılmış hematopoetik kök hücre (HSC) klonal bir hastalık var. Son zamanlarda, klonal heterojenite blast hücre nüfusunun aslında gelecek nesil sıralama (NGS) stratejileri kullanarak ve tümör hücreleri1intra klonal evrimi varlığını gösteren kurulmuştur.
Heterojenite lösemik kök hücreleri (LSC) varlığı için genişletir. Bunun üzerine lösemi ilerleme ve tedavi sırasında uygulanan çeşitli seçim baskıların üstesinden gelmek için bu dinamik hücre bölümü geliştikçe onaylanmadığına karar. Bu nedenle LSC gereken geçerli kemoterapi rejimleri için dayanıklı ve derin klinik sonuçları2ile hastalık nüks aracılık. Çeşitli işaretler LSC CD1233, CLL-14, CD975 veya TIM-36gibi karakterize etmek için tarif edilmistir. CD34 + CD38 bölmeli blast hücrelerinin LSC'yi7 için zenginleştirilmiş kabul edilir ancak normal HSC içerir. CD34 + CD38 uygulanan CD90 ve CD45RA ifade-(P6) yuvası (şekil 1) izin veren çeşitli aşamalarında normal ve malign hematopoetik öncüleri8ayırmak için. Özellikle, normal CD34 + CD38-CD90 + CD45RA-HSCs, multipotent ataları (MPP) CD34 + CD38-CD90dimCD45RA-hücreleri ve CD34 + CD38 gibi-CD90-CD45RA + multipotent progenitör (LMPP) astarlanmalıdır lenfoid hücrelerin AML çoğunluğu olarak belirlenen durumlarda8 ,9. (MFI) ortalama Floresans yoğunluğunu CD38 CD34 + hücre bölmesi içinde dikkate alınması gereken özellikle önemlidir. CD38 yoğunluk üç yeni cep bölmeleri bunların tanımladığından: CD38 negatif (P6) CD38 dim (P7) ve CD38 parlak (P8) (şekil 1). CD38 MFI bu hücreler şiddetle CD38 hızlı gibi ya hematogones ve/veya plazma hücreleri bir iç pozitif kontrol kullanarak belirlenebilir.
İmmünyetmezligi başını SALLAMAK/SCID/IL2Rγcnull (NSG) fare modeli normal engraftment için yaygın olarak kullanılır ve10,11kötü huylu insan hematopoetik hücreler. Seri xenotransplantation deneyleri deneysel LSC veya HSC işlevini doğrulamak için kullanılır. Bu çalışmalar geniş büyük ölçekli sıralama yaklaşımlar tarafından analiz edilmiştir. Yine de, daha az onun birincil AML (Şekil 2) karşılaştırıldığında NSG fareler içine engrafted AML patlama nüfus LSC ve HSC immunophenotype hakkında bilinir.
LSC sayıda böylece doğal olarak düşük, kimlik ve LSC miktar zor olan ve burada anlatılan yöntemi bir akış sitometrik tahlil tanı ve klinik takip (kalıntı LSC olabilir gibi kemoterapi yanıtı değerlendirmek için kullanılıyor olabileceği AML relapse) neden. LSC varlığı olumlu minimal rezidüel hastalık (MRD) gösterebilir. Bugüne kadar MRD AML içinde çoğunlukla izleme itimat moleküler yöntemler (Yani, RT-PCR, NGS)10,12. Ancak, bu protokol için biz aynı anda protein ifade çeşitli HSC/LSC işaretleri (CD34 CD38 CD45RA, CD90) birincil blast hücrelerinin üzerinde insan AML multiparametric Akış Sitometresi2,8,9tarafından tespit. Antikorlar bu arada herhangi bir standart Akış Sitometresi laboratuvarda uygulanabilir ve bu akışı MRD tahlil özellikle ilginç olduğunda MRD tarafından gerçek zamanlı Polimeraz zincir reaksiyonu (RT-PCR) mümkün değildir Yani, eğer lösemi özel moleküler işaretleyicileri tanılama AML örnekte algılanabilir değildir. Ayrıca, bu iletişim kuralını, daha hassas moleküler MRD teknikleri için tamamlayıcı bu tespit ve anormal hematopoetik progenitör hücre ölçmek için amaç gibi temsil eden bir işlev hücre karakteristik (şekil 3).
Akış Sitometresi ile antikor klinik laboratuvarlar çoğunda kullanılabilir tarafından üç hematopoetik progenitör nüfus ve sözde LSC yuvası ile farklı olgunlaşma derecesini ölçmek için nasıl gösterir. Ayrıca, bu hücre bölmeleri içinde karşılık gelen hasta-türetilen-xenografts (PDX) varlığını doğruladı ve tedavi takip.

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			<td>Ozyme</td>
			<td>BLE420301</td>
			<td style="width:467px;">RBC Lysis solution A : 8.29 g of NH4Cl + 0.037 g of Na2EDTA in 100 mL of sterile H2O and RBC Lysis solution B: 1 g of KHCO3 in 100 mL of sterile H2O</td>
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			<td height="20" style="height:21px;">Anti-CD90-APC (clone 5E10)</td>
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			<td height="20" style="height:21px;">Anti-CD45RA-APC-H7 (clone HI100)&#160;</td>
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			<td>560674</td>
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			<td>B92396</td>
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			<td height="20" style="height:21px;">Anti-CD123-PC7</td>
			<td>Beckman Coulter</td>
			<td>B13647</td>
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			<td height="20" style="height:21px;">Flow-Set</td>
			<td>Beckman Coulter</td>
			<td>A63492</td>
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			<td height="20" style="height:21px;">Flow-Check</td>
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			<td>Biolegend</td>
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			<td height="20" style="height:21px;">EasySep human PE selection kit</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
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			<td height="20" style="height:21px;">EasySep&#160; magnet&#160;</td>
			<td>Stem Cell Technologies</td>
			<td>18551</td>
			<td></td>
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flow cytometry to estimate leukemia stem cells in primary acute myeloid leukemia and in patient-derived-xenografts, at diagnosis and follow up

Akut Miyeloid Lösemi (AML) heterojen, olduğunu ve eğer tedavi değil, ölümcül hastalık. Erişkinlerde lösemi ilişkili ölümlerin en sık nedenidir. Başlangıçta, AML farklılaşma, lösemi blast hücrelerinin sonraki birikimi tutuklanması tarafından karakterize ve üretim fonksiyonel hematopoetik öğelerinin azaltılmış hematopoetik kök hücre (HSC) bir hastalık var. Heterojenite lösemi kök hücreleri (LSC) varlığı uzanır, bu dinamik hücre ile çeşitli seçim baskıların üstesinden gelmek için bölme gelişen üzerine lösemi ilerleme ve tedavi sırasında empoze. Daha fazla LSC nüfusu tanımlamak için CD34 + CD38 hücreli bölmenin içinde normal HSCs ve multipotent ataları ayrımcılık CD90 ve CD45RA eklenmesini sağlar. Burada, biz birkaç sözde LSC işaretleri (CD34 CD38 CD45RA, CD90) aynı anda ifade algılamak için bir protokol multiparametric Akış Sitometresi tarafından birincil blast hücrelerinin üzerinde insan AML anahat. Ayrıca, üç yaratıcı nüfus ve artan olgunlaşma derecesi ile sözde bir LSC nüfus ölçmek için nasıl gösterir. Biz bu nüfus içinde karşılık gelen hasta-türetilen-xenografts varlığını doğruladı. Bu yöntem algılama ve sözde LSC miktar kemoterapi yanıt klinik izlemek için kullanılan (i.e., minimal rezidüel hastalık), kalan LSC AML nüks neden olabilir gibi.

Burada yaratıcı nüfus normal ve kötü huylu insan kemik iliği örneklerinde belirlemek için bir yöntem mevcut. Kemik iliği ve dalak başarılı bir PDX toplanan ve multiparametric Akış Sitometresi yukarıda açıklandığı gibi yapılır. Biz birkaç blast hücreler arasında tanı hasta örnek ve karşılık gelen PDX ve özellikle, CD34 + CD38 progenitör yuvası, özellikle sözde LSC zenginleştirilmiş altgrupları göre. Biz CD34 + CD38-şok kesir PDX tanılama hasta örnek (%3,48 %0,53, <strong class="x...

Watch this Scientific Journal Video about Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up at JoVE.com

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Biz aynı anda ifade birincil akut myeloid lösemi hücreleri üzerinde lösemi kök hücre işaretlerinin Akış Sitometresi tarafından tespit etmek için bir protokol anahat. Biz üç yaratıcı nüfus ve artan olgunlaşma derecesi ile sözde bir LSC nüfus ölçmek için nasıl gösterir. Biz bu nüfus içinde karşılık gelen hasta-türetilen-xenografts varlığını doğruladı.

Akış Sitometresi lösemi kök hücre birincil Akut Miyeloid Lösemi ve hasta-türetilen-xenografts, tanı ve takip yukarı tahmin etmek için

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

The overall goal of this procedure is to estimate leukemia stem cells in acute myeloid leukemia and in patient-derived-xenografts by flow cytometry at diagnosis and at treatment follow up. This method can answer key questions in the hematology/oncology field, such as the clinical relevance of the quantitative assessment of leukemia stem cells by flow cytometry in acute myeloid leukemia. The main advantage of this technique is that it is applicable to most hematology/oncology laboratories with commonly used antibodies.This procedure will be demonstrated by Thomas, a post-doc, Fanny, a grad student, and Adeline, an engineer from my laboratory. To begin, collect two milliliters of bone marrow from de novo AML in tubes containing 1.8 milligrams per milliliter of the anti-coagulant EDTA. Isolate mononuclear cells from red blood cells and granulocytes by first diluting bone marrow in three volumes of PBS.Then, carefully overlay one volume of ficol with one volume of diluted bone marrow. Spin the density gradient at 300g for 30 minutes without a break. Then, transfer the white buffy coat layer of mononucleated cells into a new sterile tube.To remove the supernatants contaminating serum components, add 10 milliliters of PBS and centrifuge the cells at 300g for five minutes before repeating the wash. Lyse the red blood cells by adding two milliliters of lysis buffer to the cell pellet and gently vortex. Then, incubate the cells at room temperature for five minutes.Centrifuge the cells at 300g for five minutes and remove the supernatant. Then, use PBS to wash the cells. After isolating blast cells from bone marrow according to the text protocol, under a laminar flow hood, insert an unconditioned NSG mouse into a restraining device and inject five times 10 to the sixth AML blast cells into the tail vein.Return the mouse to an individual ventilated cage under conventional and specific pathogen-free conditions. Every two weeks, use a submandibular collection method with a hematocrit capillary to draw 60 microliters of blood. Transfer the capillary into a five milliliter tube then with a sterile gauze pad, apply gentle pressure to stop the bleeding.Add one milliliter of lysis buffer to the tube and gently vortex. Then, incubate the cells at room temperature for five minutes. Centrifuge the cells at 300g for five minutes.Then, after removing the supernatant, add 100 microliters of PBS with 10%BSA, three microliters of anti-human CD45 FITC, incubate for 30 minutes. Then, wash the cell pellet two times in PBS, two milliliters, with 10%BSA and centrifuge at 300g for five minutes. Every two weeks, use flow cytometry to analyze the engraftment by chimerism analysis of human versus murine CD45 expression.When the blast count is greater than 70%or severe clinical signs of sickness are observed, sacrifice the animal and collect mononuclear blast cells from a crushed spleen and from bone marrow by using PBS to flush tibias and femurs as previously described. To carry out panel staining, combine conjugated antibodies with human primary or patient-derived-xenograft, or PDX samples, and vortex the tubes. Then, incubate the cells in the dark at room temperature for 15 to 30 minutes.Remove the unbound antibodies by using two milliliters of PBS to wash the cells. Re-suspend the cells in 450 microliters of PBS, then proceed to flow cytometry analysis and MRD monitoring. To accurately and reproducibly assess membrane of cytoplasmic markers by flow cytometry, verify optical alignment.Proper tuning of the 3D system and optical sensitivity and stomatization using stomazine calibration beats every day. Use normal bone marrow to set the gates for CD38. A minimum of 500, 000 events are required and it is verified that each flow run is regular and homogenous.In addition, doublets, as well as dead cells and cell debris are eliminated. Immature hematopoietic cells are defined by being CD45 intermediate, SSC low. And the CD38 positive control or hematogones, are CD19/CD38 double positive.Then P8 or CD38 positive, P7 or CD38 intermediate, and P6 or CD38 negative are defined. The P6 cells can be further subdivided into HSC, MPP, and LSC. Since it is a normal bone marrow it is LSC negative.Using multiparametric flow cytometry, it was found that the CD34 positive, CD38 negative blast fraction was higher in PDX compared to the diagnostic patient sample. Interestingly, a higher percentage of putative LSC was found in the PDX compared to the primary patient sample, suggesting a possible enrichment of malignant progenitor cells in the hematologic tissues in this mouse model. Upon comparison of the blast progenitor populations in two different patient samples, the CD34 positive, CD38 negative fraction increased from 0.06%at diagnosis to 3.33%at the first treatment follow-up in the first patient.In contrast, the CD34 positive, CD38 negative fraction decreased in the second patient from 7.8%at diagnosis to 2.27%at the first treatment follow-up. Accordingly, the putative LSC fraction increased in the first patient from 0%at diagnosis to 0.65%and decreased in the second patient from 6.57%at diagnosis to 1.44%at treatment follow-up. Minimal residual disease or MRD monitoring, using as molecular markers the NPM1 mutation in patient one and the CBF-beta MYH11 gene fusion for patient two showed that molecular MRD decreased less in the first patient compared to the second patient for the first treatment follow-up point.Once mastered, this technique can be done in two hours if it is performed properly. While you're attempting this procedure, it is important to verify flow cytometric settings and CD38 gauging. Following this procedure, additional methods can be performed, like flow cytometric cell sorting in order to answer additional questions like functional leukemia stem cell assess.After watching this video you should have a good understanding of how to detect and quantify leukemia stem cells in patient samples in patient-derived-xenografts. Don't forget that working with patient samples can be hazardous and precautions such as GOP guidelines and institutional safety procedures should always be taken while you are performing this procedure.

Research

JoVE Journal

Cancer Research

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Örnek ve işlem kemik iliği ölçülebilir artık hastalık ve akut Myeloid lösemi lösemik kök hücre ölçmek için kapsamlı iletişim kuralı

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Kanser Araştırmaları

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

Birincil Akut lenfoblastik lösemi hücreleri hazırlanmasında farklı hücre döngüsü aşamaları santrifüj Elutriation tarafından

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

Sarkom hasta türevi xenografts tümör-başlatma hücrelerinin yalıtım ve karakterizasyonu

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Roman NFAT2 hedef genleri kronik lenfositik lösemi olarak tanımlamak için bir kromatin Immunoprecipitation tahlil

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

Birincil lösemi hücrelerinin metabolik profili değerlendirilmesi

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

Murine Hematopoetik Kök ve Progenitor Hücrelerde Mitokondriyal Reaktif Oksijen Türlerinin Akış Sitometrik Analizi ve MLL-AF9 Tahrikli Lösemi

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nükleer/Sitoplazmik Protein Ticaretini İzlemek Için Primer Kronik Lenfositik Lösemi Hücrelerinin Subsellüler Fraksiyonları

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

Yapısal DNA Alterasyon Analizi ve Hasta Kaynaklı Ksenogreftler Kullanılarak Kanserde Hedefli Tedavilerin Test Edilmesi

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Akut Miyeloid Lösemide Kök Hücreleri Toplu AltÜlmelerden Ayırt Etmek İçin NKG2D Ligand Yüzey Tespitinin İki Akış Sitometrik Yaklaşımı

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

Farelerde akut miyeloid lösemi üretmek için intra-peritoneal transplantasyon

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...