Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktailReportar como inadecuado




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BMC Biotechnology

, 8:6

First Online: 29 January 2008Received: 17 October 2007Accepted: 29 January 2008

Abstract

BackgroundHigh purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia CLL.

ResultsWe adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood PB. PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation DGC, while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC RS+DGC. The purity of CLL cells after DGC averaged 74.1% range: 15.9 – 97.4%. Using RS+DGC, the purity averaged 93.8% range: 80.4 – 99.4% with 23 of 29 79% samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis.

ConclusionThis study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.

AbbreviationsCLLChronic Lymphocytic Leukaemia

DGCdensity gradient centrifugation

FACSfluorescent-activated cell sorting

MCSmagnetic cell sorting

PBperipheral blood

PBSDulbecco-s phosphate buffered saline

PCRpolymerase chain reaction

RINRNA Integrity Number

RSRosetteSep™

RS+DGCaddition of RosetteSep B-cell enrichment cocktail prior to density gradient centrifugation

RT-PCRreverse transcription polymerase chain reaction

WBCwhite blood cell

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-8-6 contains supplementary material, which is available to authorized users.

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Autor: Salim Essakali - Dennis Carney - David Westerman - Peter Gambell - John F Seymour - Alexander Dobrovic

Fuente: https://link.springer.com/article/10.1186/1472-6750-8-6







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