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

Dette arbeidet ble delvis støttet av den franske Association Laurette Fugain, LigueContre le kreft (Nord Center), Fondation de France (leukemi Committee), SIRIC Oncolille og franske National Cancer Institute.

Vi følger europeiske standarder for bruk av dyr for vitenskaplige formål. Denne studien ble godkjent av den lokale etikk (#3097-2015120414583482v3).
1. sample forberedelse
<ol><li>Samle benmargen (2 mL) fra de novo AML i rør som inneholder antikoagulerende ethylenediaminetetraacetic syre (EDTA) i en konsentrasjon av 1,8 mg per mL av benmarg.</li>
	<li>Utføre Ficoll separasjon, en tetthet gradert separasjon isolere mononukleære celler fra røde celler og granulocytter, som fulgt...

<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>

En nøyaktig og reproduserbar vurdering av membran eller cytoplasmatiske markører av flowcytometri krever at instrument-innstillingene er bekreftet hver dag for optisk justering, riktig funksjon av det fluidic system, optisk følsomhet, og standardisering bruke kalibrering perler.
Påvisning av blast celle populasjoner i AML bruker CD90 og CD45RA multi parametrisk flyt cytometri analysen er en relativt enkel og pålitelig måte. Et viktig skritt i denne analysen er en nøyaktig vurdering av C...

Akutt myelogen leukemi (AML) er den vanligste årsaken av leukemi-assosiert dødelighet. I utgangspunktet er AML en klonal sykdom i blodkreft stamceller (HSC) preget av arrestasjonen av differensiering, etterfølgende opphopning av blast celler, og redusert produksjon av funksjonelle blodkreft elementer. Nylig er klonal heterogenitet av blast celle befolkningen etablert i hovedsak ved hjelp av neste generasjons sekvensering (NGS) strategier og viser eksistensen av intra klonal utviklingen av tumor celler1.
Heterogenitet utvider tilstedeværelsen av leukemic stamceller (Lexmarks Løsningssenter). Det er hypotesen at dette dynamiske celle kupé utvikles for å overvinne ulike utvalg press påtvunget under leukemi progresjon og behandling. Derfor Lexmarks Løsningssenter er ment å være motstandsdyktig mot gjeldende chemotherapeutic regimer og formidle sykdommen tilbakefall, med dype klinisk implikasjoner2. Ulike merker har blitt beskrevet som betegner Lexmarks Løsningssenter som CD1233, CLL-1-4, CD975 eller TIM-36. CD34 + CD38-rommet blast celleområde regnes å være beriket i Lexmarks Løsningssenter7 men også normal HSC. CD90 og CD45RA uttrykk brukt CD34 + CD38-(P6) kupé (figur 1) tillater for å skille flere stadier av normal og ondartet blodkreft forløpere8. Spesielt vanlig CD34 + CD38-CD90 + CD45RA-HSCs, multipotent progenitors (MPP) som CD34 + CD38-CD90dimCD45RA-celler og CD34 + CD38-CD90-CD45RA + lymfoide primet multipotent stamfar (LMPP) celler kan bestemmes i fleste AML tilfeller8 ,9. Mener fluorescens intensitet (MFI) CD38 er spesielt viktig vurderes i CD34 + celle batterirommet. Fordi CD38 intensitet definerer tre nye cellen avdelinger av: CD38 negative (P6), CD38 dim (P7) og CD38 lyse (P8) (figur 1). MFI CD38 bestemmes bruker enten hematogones og/eller plasma celler som en positiv internkontroll som disse cellene sterkt express CD38.
Immunodeficient hilsen/SCID/IL2Rγcnull . (NSG) musemodell er mye brukt for engraftment av normal og ondartet menneskelige blodkreft cellene10,11. Føljetong xenotransplantation analyser brukes til å validere eksperimentelt Lexmarks Løsningssenter eller HSC funksjonen. Disse studier er grundig analysert av storskala sekvensering tilnærminger. Likevel er mindre kjent om Lexmarks Løsningssenter og HSC immunophenotype AML blast befolkningen engrafted i NSG mus sammenlignet sin primære AML (figur 2).
Antall Lexmarks Løsningssenter er iboende lav dermed, identifikasjon og kvantifisering av Lexmarks Løsningssenter er utfordrende og metoden beskrevet her kan brukes som en flyt cytometric analysen diagnose og klinisk følge å evaluere kjemoterapi respons (som gjenværende Lexmarks Løsningssenter kan forårsake AML tilbakefall). Tilstedeværelsen av Lexmarks Løsningssenter kan angi positiv minimal gjenværende sykdom (MRD). Til dags dato, MRD overvåking i AML meste stole på genetiske metoder (dvs., RT PCR, NGS)10,12. Men i denne protokollen oppdage vi samtidige protein uttrykk for flere HSC/Lexmarks Løsningssenter (CD34, CD38, CD45RA, CD90) på primære blast celler av menneskelig AML ved multiparametric flyt cytometri2,8,9. Denne kombinasjonen av antistoffer kan brukes i alle standard flyt cytometri laboratorium og denne flyten MRD analysen er spesielt interessant når MRD av sanntids polymerasekjedereaksjons (RT-PCR) ikke er mulig, dvs.hvis leukemi spesifikke molekylær markører er ikke synlig i diagnostiske AML utvalget. Videre er denne protokollen, komplementære til de mer følsomme molekylær MRD teknikkene, som mål å oppdage og kvantifisere unormal blodkreft stamfar celler som representerer en funksjonell celle karakteristisk (Figur 3).
Vi viser hvordan kvantifisere tre blodkreft stamfar befolkninger og antatte Lexmarks Løsningssenter rommet med forskjellige grader av modning av flowcytometri med antistoffer tilgjengelig i fleste kliniske laboratorier. Videre vi bekreftet tilstedeværelse av cellen seksjonene i tilsvarende pasient-avledet-xenografts (PDX) og på behandling oppfølging.

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			<td style="width:189px;">PAN-Biotech</td>
			<td style="width:128px;">P04-60500</td>
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			<td height="75" style="height:75px;">RBC Lysis Buffer (10x)</td>
			<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;">NOD/SCID/IL2R&#947;cnull&#160; (NSG)&#160; mice</td>
			<td>JACKSON LABORATORY</td>
			<td>Stock No: 005557</td>
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			<td>MACS Buffer Miltenybiotec</td>
			<td>130-091-221</td>
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			<td>B36289</td>
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			<td>328114</td>
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			<td>A07770</td>
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			<td>Beckman Coulter</td>
			<td>B92417</td>
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			<td height="20" style="height:21px;">Anti-CD45RA-APC-H7 (clone HI100)&#160;</td>
			<td>Beckton Dickinson</td>
			<td>560674</td>
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			<td height="20" style="height:21px;">Anti-CD38-Pacific Blue (clone LSI98-4-3, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B92396</td>
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			<td height="20" style="height:21px;">Anti-CD45-KO (clone J.33, Iotest)</td>
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			<td>B36294</td>
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			<td>A07747</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD123-PC7</td>
			<td>Beckman Coulter</td>
			<td>B13647</td>
			<td></td>
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		<tr height="20">
			<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>
			<td>Beckman Coulter</td>
			<td>A63493</td>
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			<td height="20" style="height:21px;">Navios</td>
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			<td height="20" style="height:21px;">LSR Fortessa X20</td>
			<td>BD Biosciences</td>
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			<td height="20" style="height:21px;">CST BD</td>
			<td>BD Biosciences</td>
			<td>655051</td>
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			<td>BD Biosciences</td>
			<td>336911</td>
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			<td height="20" style="height:21px;">Anti-hCD45-FITC</td>
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			<td>BLE304006</td>
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			<td height="20" style="height:21px;">Anti-mCD45-APC</td>
			<td>Biolegend</td>
			<td>BLE103112</td>
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		<tr height="20">
			<td height="20" style="height:21px;">EasySep human PE selection kit</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;">EasySep&#160; magnet&#160;</td>
			<td>Stem Cell Technologies</td>
			<td>18551</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

Akutt myelogen leukemi (AML) er en heterogen, og hvis ikke behandles, dødelig sykdom. Det er den vanligste årsaken av leukemi-assosiert dødelighet hos voksne. I utgangspunktet er AML en sykdom i blodkreft stamceller (HSC) preget av arrestasjonen av differensiering, etterfølgende opphopning av leukemi blast celler, og redusert produksjon av funksjonelle blodkreft elementer. Heterogenitet utvider tilstedeværelsen av leukemi stamceller (Lexmarks Løsningssenter), med dynamisk cellen kupé utvikling for å overvinne ulike utvalg press påtvunget under leukemi progresjon og behandling. For å ytterligere definere befolkningen Lexmarks Løsningssenter, kan CD90 og CD45RA diskriminering av vanlig HSCs og multipotent progenitors innen CD34 + CD38 celle batterirommet. Her skissere vi en protokoll for å oppdage samtidige uttrykket av flere mulige Lexmarks Løsningssenter markører (CD34, CD38, CD45RA, CD90) på primære blast celler av menneskelig AML av multiparametric flowcytometri. Videre viser vi hvordan kvantifisere tre stamfar befolkninger og antatte Lexmarks Løsningssenter innbyggere med økende grad av modning. Vi bekreftet tilstedeværelse av disse befolkningene i tilsvarende pasient-avledet-xenografts. Denne metoden for påvisning og kvantifisering av antatte Lexmarks Løsningssenter kan brukes for klinisk oppfølging av kjemoterapi respons (dvs., minimal gjenværende sykdom), gjenværende Lexmarks Løsningssenter kan forårsake AML tilbakefall.

Her presenterer vi en metode for å bestemme stamfar populasjoner i normal og ondartet human benmarg prøver. Vi samlet benmargen og milten fra en vellykket PDX og utført multiparametric flowcytometri som beskrevet ovenfor. Vi sammenlignet flere subpopulasjoner av blast celler mellom diagnostiske pasienten prøven og den tilsvarende PDX og spesielt CD34 + CD38-stamfar rommet, særlig beriket i antatte Lexmarks Løsningssenter. Vi har funnet at CD34 + CD38-blast brøken var høyere i PDX sammenlignet med diagnostiske pas...

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

Vi skissere en protokoll for å oppdage samtidige uttrykket av leukemi stamcelleforskningen markører på primære akutt myelogen leukemi celler av flowcytometri. Vi viser hvordan kvantifisere tre stamfar befolkninger og antatte Lexmarks Løsningssenter innbyggere med økende grad av modning. Vi bekreftet tilstedeværelse av disse befolkningene i tilsvarende pasient-avledet-xenografts.

Flow cytometri å anslå leukemi stamceller i primære akutt myelogen leukemi og pasient-avledet-xenografts, diagnose og følge opp

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

Omfattende protokoll for å prøve og prosessen benmarg for å måle målbare gjenværende sykdom og Leukemic stamceller i akutt myelogen leukemi

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

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

Forberedelse av primære akutt lymfatisk leukemi celler i forskjellige cellen syklus faser av sentrifugal Elutriation

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

Isolering og karakterisering av tumor-initiere celler fra sarkom pasient-avledet Xenotransplantater

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

En Chromatin Immunoprecipitation analysen identifisere romanen NFAT2 målet gener i kronisk lymfatisk leukemi

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

Vurdering av metabolske profilen til primære leukemi celler

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

Flow analytiske analyse av mitokondrie reaktive oksygen arter i murine blodkreft stem og stamceller og MLL-AF9 drevet leukemi

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

Subcellulære Fraksjonering av primær kronisk lymfatisk leukemi celler til å overvåke kjernefysisk/cytoplasmatiske protein smugling

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

Testing av målrettede behandlinger mot kreft ved hjelp av strukturell DNA-endringsanalyse og pasientavledede Xenografts

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

To flowcytometriske tilnærminger til NKG2D Ligand overflatedeteksjon for å skille stamceller fra bulk subpopulations i akutt myeloid leukemi

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

Intraperitoneal transplantasjon for generering av akutt myelogen leukemi hos mus

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