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«3. Medizinische Klinik und Poliklinik – Hämatologische Forschung Cks1 is a critical regulator of hematopoietic stem cell cycling, quiescence and ...»

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In all transplantations 129S2xLy5.1 recipients were lethally irradiated with 9Gy (Gulmay irradiation unit). Donor cells were injected into the tail vain of the recipients. Serial whole bone marrow transplantations were performed using 2x105 donor BM cells for primary transplants and 1x106 for secondary transplants. In both cases 4x105 helper spleen cells from Ly5.1 mice were cotransplanted. 300 CD150+ FACS-sorted LSK cells were transplanted with 5x105 helper spleen cells and 1x105 BM cells delivered from mice with the same background as the recipients. For secondary transplantations 300 donorderived CD150+ LSK cells from the primary recipients were used with the same number of helper and competitor cells used in the primary transplantation.

3.4. 5-FU and BrdU injections 5-FU (Sigma-Aldrich) was diluted in HF2 Buffer and 150µg/g were injected intraperitoneally (i.p.). The mice were sacrificed 6 days after injection.

For proliferation analysis BrdU (BD Bioscience, 50µg/g body weight) was injected i.p. 12 hours before sacrificing mice.

METHODS

3.5. Colony forming assay In the colony forming assay, hematopoietic cells in low density are plated in a semi-solid medium (methylcellulose), substituted with a particular cocktail of cytokines, which allows the building of a colony from a single cell. The colony forming assay tests the progenitor number and identity as well as the clonogenetic capacity of hematopoietic cells and has been used successfully for decades [12, 168].

In this study, colony formation analyses with freshly isolated FL, BM or lineage depleted cells were performed with MethoCult M3434 (Stem Cell Technologies) methylcellulose medium. Cells were plated into methylcellulose in 250 µl IMDM (Gibco life technologies) supplemented with 20% FCS and cultured at 37°C with 5% CO2 for 12 days.

In replating experiments sorted cells were initially cultured for 12 days, and were replated each 7 days for the following analysis. The replating steps were performed by homogenizing the methylcellulose samples with blunt needles.

In case of BCR-ABL-GFP-infected BM, cells were sorted for GFP-positivity and the indicated surface markers, and plated into methyl cellulose without growth factors (MethoCult M3434, Stem cell Technologies), thus allowing proliferation of only BCR-ABL transformed cells.

3.6. Co-cultures of bone marrow cells with a stromal cell line The stromal cell line EL08-1D2, which is known to maintain hematopoietic stem and progenitor cells in vitro [163] was used for culturing of BM cells. Lineagedepleted bone marrow cells (Lin-) were co-cultured with 30 Gy irradiated confluent stromal cells. Irradiation of the stromal cells was performed in order to prevent overgrowing of the cells. The lineage fraction was negatively selected from flushed bone marrow (Lineage depletion kit, Miltenyi Biotec) using a mixture of primary biotinylated antibodies (CD5, B220, CD11b, Gr-1 and Ter119) and subsequently Streptavidin-coupled magnetic MicroBeads separation. The labeled cells were run through columns in a magnetic field.

Cells coated with MicroBeads were restrained in the column and the lineage negative, unlabeled cells were collected. 5000 Lin- cells were plated on the confluent and irradiated stromal cells in a 3 cm dish. The cells were cultured in METHODS long-term culture medium (M5300 (StemCell Technologies) with added 200 µM Glutamax (Life Technologies),100 U Penicillin (Sigma Aldrich) and 100 µg streptomycin (Life Technologies)).

3.7. Culture of bone marrow cells For culturing in vitro, freshly isolated BM cells were depleted from lineagecommited cells, using lineage cell depletion kit (Miltenyi Biotec), resuspended in MyeloCultTM 5300 (Stem cell Technologies), supplemented with SCF (100 ng/ml), TPO (20 ng/ml) and Flt3L (50 ng/ml) (all from R&D Systems) and cultivated at 37°C with 5% CO2.

The cells were than used in apoptosis assays or BrdU was added to assess the proliferation.

3.8. Cell culture All cell lines (except activation of BaF3-ts p210 at 32°C) were cultured at 37°C with 5% CO2.

The stromal cell line EL08-1D2 was cultured on 0.1 % gelatine-coated dishes in stroma medium (AlphaMEM (Life Technologies), 15 % FCS (PAA), 5 % HS (BioWhittaker), 100 U Penicillin,100 µg streptomycin (Life Technologies), 8 µM folic acid (Sigma-Aldrich), 80 µM inositol (Sigma-Aldrich), 10 µM βmercaptoethanol (Life Technologies)) with 20 % conditioned medium from the previous passage.

NX (Phoenix) Eco 293T cells were cultured in DMEM (Life Technologies) with 10 % FCS (PAA).

BaF3-p210 and BaF3-ts p210 were cultured in RPMI supplemented with 10% FCS (PAA). At 37°C, the BaF3-ts p210 cell line was additionally cultivated with IL3. To activate expression of BCR-ABL in the BaF3-ts p210 line, cells were cultivated at 32°C without IL3.

3.9. Determination of cell number and cell vitality The number of vital cells was identified using a hemocytometer and 0,5% Trypan blue solution. The cell suspension was resuspended in the Trypan blue solution in 1:1 dilution and a small drop of the mixture was applied in the METHODS hemocytometer. Dead cells absorb Trypan blue and are stained blue. Vital cells possess intact cell membranes, and exclude the dye therefore they appear in a bright white color under the microscope. The unstained cells in the four square were counted and the number of vital cells was determined by multiplication of the number per square with the dilution factor 2 and the factor 104.





3.10. Cell cycle analysis DNA synthesis and cell proliferation can be measured through integration of the thymidine-analog bromodeoxyuridine (BrdU) into the DNA of cells during S Phase progression. In order to perform full cell cycle analysis, the DNA binding dye, 7-Aminoacctinomycin (7-AAD) can be added after the BrdU staining. 7AAD labels all DNA, in this way, the different cell phases can be determined and quantitated according to the fluorescence intensity.

For in vivo analysis, BrdU was injected in the mice as described in chapter 3.4.

For in vitro labeling, cells were incubated for 1 hour at a final concentration of 10 µM BrdU. Prior BrdU detection, fresh isolated or cultured bone marrow cells were stained for surface markers and subsequently fixed and permeabilized. To assess the incorporation of BrdU in the DNA, a fluorochrome-conjugated (FITC or APC) antibody against BrdU was used. The BrdU staining was performed following the manufacturer’s instructions (BrdU Flow Kit, BD Biosciences).

3.11. Apoptosis assay Apoptosis in early stages at a cellular membrane level is characterized through phosphatidyl serine molecules (PS) flipping [169]. In healthy cells, PS are located in the inner layer of the cell membrane. At the early stage of apoptosis PS flip on the outer layer of the cell membrane and serve as a signal for macrophages to eliminate the apoptotic cell at later stage [169]. Annexin VFITC (BioLegend) is a protein dye with strong affinity to PS in the presence of Ca2+. Once attached to PS of apoptotic cells, the cells stay positive with Annexin V-FITC and can be detected with FACS.

In order to induce apoptosis, cultivated BM cells were washed from the supplemented factors and incubated for 24 hours. The cells were then stained as described in 3.13. After the last washing step, however, cells were METHODS resuspended in 500 µl annexin buffer (10 mM HEPES pH 7.4 (Life Technologies), 140 mM NaCl (Carl Roth), 2.5 mM CaCl2 (Sigma-Aldrich)) with added Annexin V-FITC (BioLegend) and PI. The cells were incubated at 4°C for 15 min before measurement.

3.12. Retroviral transduction In this project BM cells were transduced with p210-BCR-ABL to be used in colony forming assays. The retroviral packaging cell line Phoenix-Eco-293T (Phoenix-E) was transfected with the MSCV-p210-GFP vector. The virus was collected and used for retroviral transduction of 5-FU-mobilized BM cells.

Transfection Transfection is the process of introducing exogenous DNA into eukaryotic cells.

Phoenix-E cells were transfected using the cationic liposome substance, LipofectamineTM 2000 (Life technologies). In this method, the genetic material is introduced into the cells through liposomes. The used cationic lipids build a compact structure with the negatively charged DNA molecules. Because of their cationic charge on the outside and their lipophilic structure, these complexes interact with the negatively charged, hydrophobic cell membrane and enter the cells trough phagocytosis. For this purpose, at the day before the transfection 2,2x106 Phoenix-E cells were plated into a 6 cm dish. 10 µg DNA was used to transfect one dish of Phoenix-E cells. DNA and Lipofectamine 2000 master mix was prepared in a serum reduced medium Opti-MEM® (Life technologies) according to the manufacturer’s instructions. Six hours after the transfection procedure, the medium of the Phoenix-E cells was changed with fresh culture medium.

Collecting of the retrovirus Transfection of the Phoenix-E cell line provides the cells with the information needed to express the structural genes needed to form an infectious virus particle. The introduced DNA is transcribed to complementary RNA, packed into the virus particles and released into the medium. To generate the retrovirus the supernatant from the transfected cells was collected, filtered through 0,45 µm cell filters to exclude eventually detached Phoenix-E cells and the medium was METHODS changed with fresh culture medium. This process was repeated 3 times at every twelfth hour.

Retroviral transduction Retroviral transduction is a process, in which target cells are infected with DNA constructs by using retroviruses. Characteristic for retroviruses is the enzyme reverse transcriptase, which transcribes the virus RNA into DNA. The DNA can then be integrated into the genome of dividing cells. Primary BM material was prepared for the infection by injecting the mice with 5-FU (described in chaper 3.4.) 4 days before collecting the BM. 5-FU kills actively cycling cells, thus ablating the progenitor pool and all dividing hematopoietic cells but sparing the pool of quiescent, non-dividing HSC [170, 171]. The HSC were activated to divide and replenish the hematopoietic system. 4 days after 5-FU injection, the BM is rich in progenitor dividing cells, which are then cultivated with cytokines to further stimulate the cell division. For retroviral transduction, about 5x105 BM cells were resuspended in 500 µl culture medium and 2 ml retrovirus with added Polybrene (4µg/ml). Polybrene is a cationic polymer and serves to improve the transduction efficiency. The cells were centrifuged for 90 min at 2400 rpm at 32°C and then incubated for 12 hours at 37°C. This process was repeated four times. The transfection efficiency was determined by FACS.

3.13. Cell sorting and surface staining for flow cytometry For staining of surface markers (antibodies listed in chapter 2.5, table 1) the cells were resuspended in 100 µl staining buffer (0.5 % BSA (Sigma-Aldrich) in PBS (Life Technologies)) with primary antibodies. For the staining of mature populations, 1x106 cells were used, for HSC/HPC stains and BrdU analysis 5x106 cells were stained. The cells were incubated with the antibodies for 15 min at 4°C and after that washed with staining buffer. If necessary, a secondary antibody was added (also diluted in staining buffer, 0.5 µl antibody per 1x106 cells). The cells were incubated again for 15 min at 4°C and then washed with staining buffer. Finally, the stained cells were resuspended in staining buffer with 1 µg/ml PI (Life Technologies) and analyzed on CyAn ADP Lx P8 (CoulterCytomation). Cell sorting was performed using the MoFlo legacy 14 colour cell sorter (Beckman Coulter).

METHODS

3.14. Intracellular staining for flow cytometry For intracellular staining, freshly isolated BM cells were first depleted from lineage cells, using lineage cell depletion kit (Miltenyi Biotec), than stained for surface markers (as explained in 3. 13). Additionally cells were fixed and permeabilized using the FIX & PERM cell permeabilization reagents (Life Technologies) according to the manufacture’s instrucions and subsequently stained with antibodies for intracellular markers (chapter 2.5, table 2).

3.15. Plasmid amplification Bacterial cells, treated chemically or physically to obtain permeable cell walls are called competent bacteria. These cells are able to include exogenous circular DNA better than conventional bacteria. Competent bacteria are stored at -80°C and thawed slowly on ice prior use. In this project, One Shot TOP10 Chemically Competent E. coli (Life Technologies) were used.

In order to amplify DNA-vectors, competent bacteria were transfected with the requested plasmid, incubated over-night and the plasmid DNA was extracted using Maxi-preparation.

Transformation is the process of uptake, incorporation and expression of exogenous genetic material into bacterial cells. For this purpose, the plasmid DNA is mixed with the bacterial suspension and incubated on ice for 30 min.

Next, the bacteria are exposed to a heat shock for 30 sec at 42°C. The abrupt heating enables entering of the plasmid DNA into the cells. Immediately after the heat shock the bacteria are put on ice for 2 min. Than the cells are plated on ampicillin culture plates (1,5% Bactoagar in LB-Medium, autoclaved; ampicillin 50 mg/ml) and incubated for 12 hours at 37°C. The antibiotic in the medium serves for selective growth of only those bacteria which were successfully transfected with the DNA plasmid, containing an ampicillin resistance gene.

After twelve hours, one colony is picked and seeded into a fluid culture (1% Bacto-Trypton; 1% NaCl; 0,5% yeast extract, autoclaved; ampicillin 50 mg/ml, pH 7,0) Culturing of the cells overnight (37°C, shaking with 250 upm) amplifies the bacteria, hence the transfected plasmid DNA.

The target plasmid DNA was isolated using the Qiagen® HiSpeed® Plasmid Maxi Kit (Qiagen) according to the manufacturer’s instruction

METHODS



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