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- Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail

Peripheral blood (PB) samples of CLL patients were obtained as part of a study approved by the Peter MacCallum Ethics of Human Research Committee. The white blood cell (WBC) count ranged from 7.81 to 437.08 × 106/ml and averaged 76.1 × 106/ml. The diagnosis of CLL was based on earlier examination of the patients' blood film and immunophenotyping for CD3, CD4, CD5, CD8, CD10, CD16, CD19, CD20, CD22, CD23, CD38, CD45, CD56, FMC7 and surface immunoglobulin light chain expression.

Blood from CLL patients was incubated with RosetteSep™ (StemCell Technologies Inc., Vancouver, British Columbia, Canada) (RS) at a concentration of 70 μl/ml PB in the dark at room temperature for 20 minutes with gentle manual swirling every 5 minutes. As a control, an aliquot of the same sample was processed the same way except without addition of RS. After incubation, both aliquots of blood were diluted with 4 volumes (rather than 2 volumes as recommended by the manufacturer) of Dulbecco's phosphate buffered saline (PBS) containing 2% foetal bovine serum as otherwise we found that blood samples with high WBC counts were insufficiently diluted for efficient separation. The samples were then underlaid with 3 ml Lymphocyte Separation Medium (MP Biomedicals, Aurora, OH) and centrifuged for 20 minutes at 1,200 g. The enriched cells were subsequently harvested from the interface and washed once in 2 volumes Dulbecco's PBS with 2% foetal bovine serum by centrifuging for 10 minutes at 200 g.

The purity of the enriched cell population was analysed by staining with a panel of fluorescently labelled antibodies (BD Biosciences, San Jose, CA) in three different tubes. (Tube 1: CD5-FITC, CD10-PE, CD19-PerCP and CD45-APC. Tube 2: FMC7-FITC, CD23-PE, CD19-PerCP and CD45-APC. Tube 3: CD22-FITC, CD38-PE, CD20-PerCP and CD45-APC). Data acquisition was carried out using a FACScalibur-cytometer (BD Biosciences) and Cell Quest software (BD Biosciences). Ungated data analysis was conducted using Cytomics RXP software (Beckman Coulter, Fullerton, CA).

The diagnosis of CLL was reconfirmed by assessing positivity for CD5, CD19, CD23 and CD45, weak positivity for CD20; weak positivity or negativity for CD22 and FMC7 and negativity for CD10. After confirming the CLL diagnosis, the CLL purity was assessed by the co-expression of CD5 and CD19.

RNA was extracted from RS+DGC enriched CLL cells by applying a combination of the Trizol-protocol (Invitrogen, Carlsbad, CA, USA) and the RNeasy Micro Kit (Qiagen, Hilden, Germany). The RNA Integrity Number (RIN) of the extracted RNA was determined using the 2100 Bioanalyzer (Agilent, Santa Clara, CA) according to the manufacturer's protocol.

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