Name: colony forming cell (cfc) assay for human hematopoietic cells
Uploaded: 2010-12-18T17:00:00
Description: Watch this Scientific Journal Video about Colony Forming Cell CFC Assay for Human Hematopoietic Cells at JoVE.com

Wij danken de Alvin J. Siteman Cancer Center aan de Washington University School of Medicine en Barnes-Jewish Hospital in St. Louis, MO, voor het gebruik van de High Speed ​​Cell Sorter Core, die flow cytometrie apparatuur en sorteren van diensten. Dit werk werd ondersteund door National Institutes of Health Subsidies R01 HL082549 en K02 HL084179 (NRY). De Siteman Cancer Center wordt gedeeltelijk ondersteund door een NCI Cancer Center Support Grant P30 CA91842.

1. Bereiding van reagentia <ol><li> Bereid steriele 1000x stamoplossingen van FMS-gerelateerde tyrosine kinase 3 (FLT-3)-ligand, granulocyt / macrofaag kolonie-stimulerende factor (GM-CSF), stamcel factor (SCF), trombopoëtine (TPO), interleukine (IL) -3 , en IL-6 volgens de instructies van de fabrikant. Bereid steriele Retronectin stamoplossing (1mg/ml) ook volgens de instructies van de fabrikant. Verdeel deze voorraad oplossingen in kleine porties en bewaar bij -20 ° C tot herhaalde vries-dooi te vermijden. </li><l...

<ol>
	<li>Takeda, A., Goolsby, C., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98-HOXA9+induces+long-term+proliferation+and+blocks+differentiation+of+primary+human+CD34++hematopoietic+cells.">NUP98-HOXA9 induces long-term proliferation and blocks differentiation of primary human CD34+ hematopoietic cells.</a> Cancer Res. , 66-6628 (2006).</li><li>Yassin, E. R., Abdul-Nabi, A. M., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+NUP98-DDX10+oncogene+on+primary+human+CD34++cells:+role+of+a+conserved+helicase+motif.">Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: role of a conserved helicase motif.</a> Leukemia. , (2010).</li><li>Yassin, E. R., Sarma, N. J., Abdul-Nabi, A. M., Dombrowski, J., Han, Y., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+transformation+of+primary+human+hematopoietic+cells+by+the+oncogene+NUP98-HOXA9.">Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.</a> PLoS One. 4, e6719-e6719 (2009).</li><li>Nissen-Druey, C., Tichelli, A., Meyer-Monard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hematopoietic+colonies+in+health+and+disease.">Human hematopoietic colonies in health and disease.</a> Acta Haematol. 113, 5-96 (2005).</li><li>Pereira, C., Clarke, E., Damen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cell+assays.">Hematopoietic colony-forming cell assays.</a> Methods Mol Biol. 407, 177-208 (2007).</li><li>Coulombel, L. <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+hematopoietic+stem/progenitor+cells:+strength+and+drawbacks+of+functional+assays.">Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.</a> Oncogene. 23, 7210-7222 (2004).</li><li>Hogge, D. E., Lansdorp, P. M., Reid, D., Gerhard, B., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection,+maintenance,+and+differentiation+of+primitive+human+hematopoietic+cells+in+cultures+containing+murine+fibroblasts+engineered+to+produce+human+steel+factor,+interleukin-3,+and+granulocyte+colony-stimulating+factor.">Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor.</a> Blood. 88, 3765-3773 (1996).</li></ol>

De CFK&#39;s test is uitgebreid gebruikt om de proliferatie en differentiatie van hematopoietische voorlopercellen patronen te bepalen en om de effecten van oncogenen (4, 5) te bestuderen. Het heeft het voordeel ten opzichte van vloeibare culturen van het zijn een klonale test, zodanig dat de kolonies de nakomelingen van een enkele progenitor vertegenwoordigen en kan afzonderlijk worden verwijderd voor verdere analyse. De beperking van de CFK-test is dat het niet voldoende is voor de detectie van meer onrijpe voorloperc...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>12440</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>06150</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin (PS)</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>FLT-3 ligand</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-19</td>
<td>&nbsp;</td>
<tr>
<td>GM-CSF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-03</td>
<td>&nbsp;</td>
<tr>
<td>SCF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-07</td>
<td>&nbsp;</td>
<tr>
<td>TPO</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-18</td>
<td>&nbsp;</td>
<tr>
<td>IL3</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-03</td>
<td>&nbsp;</td>
<tr>
<td>IL6</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-06</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP118</td>
<td>&nbsp;</td>
<tr>
<td>Retronectin</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>T100A</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14200</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue solution (0.4%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
<tr>
<td>Methocult GF+ H4435</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>04445</td>
<td>&nbsp;</td>
<tr>
<td>Human Methylcellulose Enriched Media</td>
<td>&nbsp;</td>
<td>R &amp; D Systems</td>
<td>HSC005</td>
<td>&nbsp;</td>
<tr>
<td>Wright/ Giemsa stain</td>
<td>&nbsp;</td>
<td>Harleco</td>
<td>64571</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Solution, pH 6.4 - Giordano formula</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>1450</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A412-4</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 60</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>8310</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum </td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals</td>
<td>D208</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD11b phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555388</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD33 allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551378</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD71 phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555537</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD235a allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551336</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>24-well non-treated tissue culture plates</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-1147</td>
<td>&nbsp;</td>
<tr>
<td>30 mm non-treated dish</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27150 </td>
<td>&nbsp;</td>
<tr>
<td>100 mm tissue culture dish </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27500</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2097</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2070</td>
<td>&nbsp;</td>
<tr>
<td>Syringes 3 ml </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28240</td>
<td>&nbsp;</td>
<tr>
<td>16 gauge blunt-end, 1&frac12; inch needle </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28110</td>
<td>&nbsp;</td>
<tr>
<td>50 &mu;m CellTrics cell filter</td>
<td>&nbsp;</td>
<td>Partec</td>
<td>04-004-2327</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0267110</td>
<td>&nbsp;</td>
<tr>
<td>TPX sample chambers</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>A78710018</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Superfrost/Plus Microscope Slides, Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>Shandon filter cards</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>5991022</td>
<td>&nbsp;</td>
<tr>
<td>Shandon cytospin slide holder</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>59920063</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Complete Staining Assembly 100</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>100</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark Corporation</td>
<td>34155</td>
<td>&nbsp;</td>
<tr>
<td>1 &mu;m filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28307-134</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Diaphot</td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>DS-F11</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX51 </td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DP71</td>
<td>&nbsp;</td>
<tr>
<td>Scanner</td>
<td>&nbsp;</td>
<td>Microtek</td>
<td>Scanmaker 4</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-812</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator</td>
<td>&nbsp;</td>
<td>Sanyo</td>
<td>MCO-18AIC</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>&nbsp;</td>
<td>Shandon</td>
<td>Cytospin 2</td>
<td>&nbsp;</td>
<tr>
<td>Bench-top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5810-R</td>
<td>&nbsp;</td>
<tr>
<td>Water purification system</td>
<td>&nbsp;</td>
<td>Barnstead</td>
<td>Nanopure-Diamond</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

colony forming cell (cfc) assay for human hematopoietic cells

Humane hematopoietische stamcellen / progenitorcellen worden meestal verkregen uit beenmerg, navelstrengbloed of perifeer bloed en worden gebruikt om hematopoiese en leukemie te bestuderen. Ze hebben het vermogen om te differentiëren in de lymfoïde en myeloïde geslachten. De kolonievormende cel (CFC) test wordt gebruikt om de proliferatie en differentiatie patroon van hematopoietische voorlopercellen door hun vermogen om kolonies vormen in een halfvaste medium te bestuderen. Het aantal en de morfologie van de kolonies gevormd door een vast aantal van de input-cellen geven voorlopige informatie over het vermogen van voorouders om te differentiëren en zich vermenigvuldigen. Cellen kunnen worden geoogst uit afzonderlijke kolonies of van de hele plaat verder te beoordelen hun aantallen en differentiatie staten met behulp van flowcytometrie en morfologische evaluatie van de Giemsa-gekleurde dia&#39;s. Deze test is nuttig voor de beoordeling van myeloïde maar niet lymfoïde differentiatie. De term myeloïde in deze context wordt gebruikt in ruimere zin te granulocytaire, monocytische, erytroïde, en megakaryocytaire lijnen omvatten. We hebben gebruik gemaakt van deze test om de effecten van oncogenen te beoordelen op de differentiatie van primaire menselijke CD34 + cellen afkomstig uit het perifere bloed. Voor dit doel cellen getransduceerd met ofwel controle retrovirale construct of een constructie uiting van de oncogen van belang, in dit geval NUP98-HOXA9. Wij werken met een algemeen gebruikte retrovirale vector, MSCV-IRES-GFP, dat een bicistronische mRNA dat het gen van interesse en een GFP marker produceert uitdrukt. Cellen zijn vooraf geactiveerd door te groeien in de aanwezigheid van cytokinen voor twee dagen voorafgaand aan de retrovirale transductie. Na nog twee dagen, zijn GFP + cellen geïsoleerd door middel van fluorescentie-geactiveerde cel sortering (FACS) en gemengd met een methylcellulose-bevattend halfvaste medium aangevuld met cytokines en geïncubeerd tot kolonies op de oppervlakte, meestal 14 dagen. Het aantal en de morfologie van de kolonies zijn gedocumenteerd. Cellen worden vervolgens verwijderd van de platen, gewassen, geteld, en onderworpen aan flowcytometrie en morfologische onderzoek. Flowcytometrie met antilichamen die specifiek zijn voor het celoppervlak markers die tijdens hematopoiese geeft informatie over de afstamming en rijping podium. Morfologische studies van individuele cellen onder een microscoop na Wright-Giemsa kleuring nadere informatie met betrekking tot afstamming en rijping. Vergelijking van cellen getransduceerd met controle lege vector die getransduceerd met een oncogen onthult de effecten van het oncogen op hematopoietische differentiatie.

Watch this Scientific Journal Video about Colony Forming Cell CFC Assay for Human Hematopoietic Cells at JoVE.com

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

De kolonievormende cel (CFC) assay is een in vitro assay waarin hematopoietische voorlopercellen vormen kolonies in een semi-vast medium. Een combinatie van kolonie morfologie, celmorfologie en flowcytometrie worden gebruikt om het vermogen van de voorouders te laten groeien en differentiëren langs de verschillende hematopoietische lijnen te beoordelen.

Colony Forming Cell (CFC) test voor humane hematopoietische cellen

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

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In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...