Cell Sorting Facility

Dipl.-Ing. Bonaventure Ndikung Soh Bejeng
Heinrich-Heine-Universität Düsseldorf
Laboratory for Molecular Stem Cell Biology
Building 14.80, Room 03.16
Phone: +49-211-8119114
E-mail: Bonaventure.Ndikung@uni-duesseldorf.de

Research Interests

Concordant with the research carried out in the Laboratory for Molecular Stem Cell biology, based on the analysis of hematopoietic stem cells and the differentiation of hematopoietic lineages especially lymphocytes, my research interests lie on the one hand in chronic myelogenous leukemia disease progression and on the other hand in the usage (operation) of a FACStar Sorter for the effective purification of cell populations.
Chronic myelogenous leukemia is a tri-phasic clonal myeloproliferative disorder of hematopoietic stem cells that evolves from a chronic stage, through an accelerated to a blast crisis stage (Calabretta et al., 2004). An important attribute of most CML cells is the presence of an oncogenic BCR-ABL1 gene rearrangement, that results from the t(9;22)(q34;q11) translocation. This juxtaposition of the regulatory and 5’ gene segments of BCR with the 3’ catalytic domains of c-ABL leads to a chromosome known as the Philadelphia chromosome (Ph) (Lindner et al., 2003).
The proteins encoded by the BCR-ABL1 gene rearrangement are dependent on the breakpoint locations in the BCR gene. A rearrangement within the major breakpoint cluster region (MBCR) of the BCR gene forms a fusion protein of 210 kD, referred to as P210, which is found in almost all CML cells (99 percent, Laurent et al.,2001,) and in some acute lymphoblastic leukemia cells (Telegeev et al.,2004). About 70 percent of BCR-ABL1 B cell lineage acute lymphoblastic leukemia cells carry BCR-ABL1 fusion genes rearranged within the minor breakpoint cluster region (mBCR) encoding a smaller BCR-ABL1 fusion protein of 190 kD (Laurent et al.,2001). A third protein of 230 kD, P230, has also been found in chronic neutrophilic leukemia (CNL) (Martinelli et al., 2002).
Cases of CML in blast crisis exhibit a myeloid phenotype as well as a B lymphoid phenotype, the so-called ’B lymphoid blast crisis’, which provokes the question, whether they both share a common clonal origin in the chronic phase CML or if the B lymphoid blast crisis is a progeny of a second tumor clone independent of the chronic phase CML.
My aim is to acquire, with the help of cell sorting, PCR and sequencing of PCR products, a tangible understanding of CML disease progression.

References

Calabretta B, Perrotti D. The biology of CML blast crisis. Blood. 2004;103:4010-22.

Laurent E, Talpaz M, Kantarjian H, Kurzrock R. The BCR gene and philadelphia chromosome-positive leukemogenesis. Cancer Res. 2001;61:2343-55.

Lindner I, Kharfan-Dabaja MA, Ayala E, Kolonias D, Carlson LM, Beazer-Barclay Y, Scherf U, Hnatyszyn JH, Lee KP. Induced dendritic cell differentiation of chronic myeloid leukemia blasts is associated with down-regulation of BCR-ABL. J Immunol. 2003;171:1780-91.

Martinelli G, Amabile M, Giannini B, Terragna C, Ottaviani E, Soverini S, Saglio G, Rosti G, Baccarani M. Novel types of bcr-abl transcript with breakpoints in BCR exon 8 found in Philadelphia positive patients with typical chronic myeloid leukemia retain the sequence encoding for the DBL- and CDC24 homology domains but not the pleckstrin homology one. Haematologica. 2002;87:688-94.

Telegeev GD, Dubrovska AN, Dybkov MV, Maliuta SS. Influence of BCR/ABL fusion proteins on the course of Ph leukemias. Acta Biochim Pol. 2004;51:845-9.

Core Facility

The Cell Sorter facility under my auspices is a FACStar Plus from BD Biosciences, designed for efficient cellular analysis and sorting of subpopulations of interest. This is done in accordance with the principles of flow cytometry.
The FACStar Plus Cell Sorter comprises of:

  • an optical bench/sensor module with a laser, an optical system and fluidic assemblies
  • an electronics console containing all power supplies, signal processing electronics, sort electronics, indicators for operation and units for instrument setup and monitoring of analytical parameters
  • a computer system for instrument setup, control, data acquisition and data analysis.

Before sorting, cells are stained with the desired surface or nuclear markers with which the different cell populations can be identified. Tantamount to flow cytometry, cells in the sorter can be identified and defined by the following characteristics:

  • relative size (forward scatter, FSC); measured in the same axis with the laser stream
  • relative granularity and internal complexity (side scatter, SSC); measured from refracted and reflected light at 90° to the laser stream
  • as well as their specific fluorescence (FL1, FL2, FL3) and fluorescence intensity; measured in the direction of the SSC.

Cells are transported from the sample tube with help of air pressure and propelled through a carrier fluid, by hydrodynamic focusing, and a sample valve assembly to the nozzle. The stream issuing from the nozzle is a sheath fluid stream containing the sample stream with the cell suspension confined to the centre. The stream is then interrogated by the laser beam before being divided into droplets by ultrasonic frequency vibrations of the nozzle assembly. Thereafter the droplets pass the deflection plates, where the desired cells are sorted into sample collection tubes, while the sheath fluid and undesired cells are channeled into the centre stream aspirator.
Using this technical platform, we can provide cell purification services to the scientific community of the Düsseldorf campus in the following formats:

  • single cell sort
  • population sort for molecular biology or protein analysis
  • population sort for cell culture purposes (sterile conditions).

Inquiries about technical details, costs and availability should be addressed to:

Dipl.-Ing. Bonaventure Ndikung Soh Bejeng
Heinrich-Heine-Universität Düsseldorf
Laboratory for Molecular Stem Cell Biology
Building 14.80, Room 03.16
Phone: +49-211-8119114

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