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| 3MM Whatman paper |
| Whatman, Dassel, Germany |
| Autoclave |
| Integra Biosciences, Baar, Switzland |
| Benchtop gyroory incubator |
| New Brunswick Scientific, Edison |
| shaker for bacterium |
| N.J. USA |
| Bacteria incubator |
| Heraeus, Hanau, Germany |
| Bandelin Sonopuls HD 2070 |
| South Yorkshire, England |
| Cell culture flasks/dishes |
| Greiner, Heidelberg, Germany |
| Cell culture incubator |
| Heraeus, Hanau, Germany |
| CELL locate glass cover slips with grids, |
| Eppendorf, Hamburg, Germany |
| – Grid size 55 μm |
| Cell scraper |
| Corning, Wiesbaden, Germany |
| Cell strainer (40μm) |
| Becton Dickinson, Heidelberg, Germany |
| FACS (Fluorescence-activated cell sorting) |
| FACScan, Beckton Dickinson, Heidelberg |
| Centrifuge |
| Cytocentrifuge |
| Thermo Shandon, Pittsburgh, USA |
| Rotina 48 RS Table top centrifuge |
| Hettich, Tuttlingen, Germany |
| -Sorvall RC- 5B refrigerated superspeed |
| centifuge and Biofuge 13R |
| Heraeus, Hanau, Germany |
| Table top centrifuge 5415C |
| Eppendorf, Hamburg, Germany |
| Megafuge 1.0 with Rotor BS4402/A |
| Heraeus, Hanau, Germany |
| Chemiluminescence detection |
| -Film BioMax |
| Eastman-Kodak, Rochester, USA |
| Corning 125 pH meter |
| Fisher Scientific, Shwerte, Germany |
| Cryotubes |
| Nalgene, Rochester, NY , USA |
| Cytofunnel |
| Shandon, Pittsburgh, USA |
| Electroporation: |
| Gene Pulser System with electroporation |
| Bio-Rad Laboratories, Munich, Germany |
| Cuvette 0.4 cm |
| Flow Chamber Kit |
| -Circular parallel plate |
| GlycoTech, Maryland, U.S.A |
| Gel electrophoresis system |
| -DNA-sub cell and Mini-Sub cell System |
| Gibco, Betheseida, USA |
| Power PAC 2000 |
| BioRad, Munich, Germany |
| Irradiation of mice |
| -Betatron 500A |
| Siemens, Munich, Germany |
| Lumat LB 9507 |
| Berthold, Bad Wildbad, Germany |
| Magnetic cell separation |
| -MiniMACS separator |
| Miltenyi, Bergisch Gladbach, Germany |
| -MidiMACS separator |
| Miltenyi, Bergisch Gladbach, Germany |
| -MACS separation columns: LD and LS |
| Miltenyi, Bergisch Gladbach, Germany |
| Microscope |
| -SZ40 Zoom Stereo Microscope |
| Olympus, Munich, Germany |
| Axioplan II-imaging Fluorescence microscope |
| Zeiss, Goettingen, Germany |
| Leitz Labovert inverted Microscope |
| Leitz, Wetzlar, Germany |
| Zeiss ID03 inverted microscope |
| Zeiss, Goettingen, Germany |
| Nitrocellulose membrane (0.45 μm) |
| Bio-Rad Laboratories, Munich, Germany |
| PCR |
| -Techne TC-312 |
| Kisker , Steinfurt, Germany |
| Protein transfer |
| -Trans-Blot SD Semi-dry Transfer cell |
| BioRad, Munich, Germany |
| Spectrohotometer |
| -Gene Quant II |
| Pharmacia Biotech, Freiburg, |
| -Ultraspec® 2000 |
| Germany |
| Steril bank |
| Heraeus, Hanau, Germany |
| Steril filter |
| -0.22μM |
| Millipore, Eschborn, Germany |
| -0.45μM |
| Millipore, Eschborn, Germany |
| Vortex |
| Eppendorf, Hamburg, Germany |
| Water bath |
| Julabo Labortechnik, Seelbach, Germany |
| Acetic acid glacia |
| Sigma, Steinheim, Germany |
| Acetone |
| Roth, Karlsruhe, Germany |
| Ammonium persulfate |
| Fluky, Deisenhofen, Germany |
| Ampicillin |
| Sigma, Steinheim, Germany |
| Anti-Sca-1 Microbeads |
| Miltenyi, Bergisch Gladbach, Germany |
| Biorad protein dye |
| Biorad, Munich, Germany |
| Borax |
| Sigma, Steinheim, Germany |
| BrdU |
| Sigma, Steinheim, Germany |
| β-Mercaptoethanol |
| Fluka , Deisenhofen, Germany |
| Bromophenol blue |
| Sigma, Steinheim, Germany |
| Calcium chloride |
| Sigma, Steinheim, Germany |
| Chlorofor |
| Fluka , Deisenhofen, Germany |
| Cumaric acid |
| Sigma, Steinheim, Germany |
| ddH2O |
| Sigma, Steinheim, Germany |
| DEPC |
| Sigma, Steinheim, Germany |
| DMSO |
| Sigma, Steinheim, Germany |
| EDTA, disodium, dihydrate |
| Sigma, Steinheim, Germany |
| Ethanol |
| Merck, Darmstadt, Germany |
| Ethidiumbromid |
| Sigma, Steinheim, Germany |
| Formaldehyd |
| Merck, Darmstadt, Germany |
| Forene (Isofluran) |
| Abbott GmbH, Wiesbaden, Germany |
| Glucose |
| Sigma, Steinheim, Germany |
| Glutathione sepharose |
| TM |
| Amersham/Pharmacia Biotech, Freiburg, |
| Germany |
| Glycerol |
| Sigma, Steinheim, Germany |
| Glycin |
| Merck, Darmstadt, Germany |
| Isopropylthio-β-D-Galactoside (IPTG) |
| Sigma, Steinheim, Germany |
| HCL |
| Merck, Darmstadt, Germany |
| Isobutanol |
| Sigma, Steinheim, Germany |
| Isopropanol |
| Fluka , Deisenhofen, Germany |
| Kanamycin |
| Sigma, Steinheim, Germany |
| Lineage cell depletion kit |
| Miltenyi, Bergisch Gladbach, Germany |
| Luminol |
| Fluka , Deisenhofen, Germany |
| Sigma, Steinheim, Germany |
| MgSO4 |
| Fluka , Deisenhofen, Germany |
| NaN3 |
| Sigma, Steinheim, Germany |
| NaCl |
| Merck, Darmstadt, Germany |
| NaH2PO4 |
| Sigma, Steinheim, Germany |
| NaOH |
| Roth GmbH, Karlsruhe, Germany |
| Natriumacetat |
| Fluka , Deisenhofen, Germany |
| Paraformaldehyd |
| Merck, Darmstadt, Germany |
| p-Coumaric acid |
| Sigma, Steinheim, Germany |
| PD-10 columns |
| Amersham/Pharmacia Biotec, Freiburg, |
| Germany |
| Phenol |
| Fluka , Deisenhofen, Germany |
| PKH26 Red fluorescence kit |
| Sigma, Steinheim, Germany |
| Polybrene |
| Sigma, Steinheim, Germany |
| Ponceau S |
| Sigma, Steinheim, Germany |
| Propedium iodide |
| Sigma, Steinheim, Germany |
| Protease-inhibitors |
| Roche Basel, Switzerland |
| Pre-stained SDS-PAGE standards |
| -Broad range |
| Bio-Rad Laboratories, Munich, Germany |
| Retronectin |
| ® |
| Takara Bio Inc., Otsu, Japan |
| Sca-1 Isolation Kit |
| Miltenyi, Bergisch Gladbach, Germany |
| SDS |
| Sigma, Steinheim, Germany |
| STREPtactin sepharose |
| TM |
| IBA, Goettingen, Germany |
| Protein elution buffer |
| IBA, Goettingen, Germany |
| TEMED |
| Sigma, Steinheim, Germany |
| Tritriplex III, 1,1% |
| University teaching hospital Frankfurt |
| Trizma HCl |
| Sigma, Steinheim, Germany |
| Triton X-100 |
| Sigma, Steinheim, Germany |
| Tween20 |
| Sigma, Steinheim, Germany |
| Vivapore (For concentrating protein) |
| VivaScience, Stonehouse, UK |
| Xylencyanol |
| Sigma, Steinheim, Germany |
| 7-aminoactinomycin D (7-AAD) |
| Sigma, Steinheim, Germany |
| BSA, V solution |
| Sigma, Steinheim, Germany |
| Chloroquine |
| Sigma, Steinheim, Germany |
| DMEM, -High glucose (4.5g/l) |
| Invitrogen, Karlsruhe, Germany |
| DMSO |
| Sigma, Steinheim, Germany |
| FBS |
| Gibco BRL, Paisley, Schottland |
| Ficoll separating solution |
| Biochrom (# L-6115), Berlin, Germany |
| Gelatin |
| Sigma, Steinheim, Germany |
| GNF-2 |
| Sigma, Steinheim, Germany |
| HBSS |
| Gibco, Karlsruhe, Germany |
| HEPES solution |
| Gibco, Karlsruhe, Germany |
| Horse serum |
| Gibco, Karlsruhe, Germany |
| Imatinib |
| Novartis, Basel Switzerland |
| IMD |
| Gibco, Karlsruhe, Germany |
| L-Glutamine |
| Gibco, Karlsruhe, Germany |
| Methoculttm GF M3434 |
| StemCell Technologies, Vancouver, |
| Canada |
| Mycosa |
| Gibco, Karlsruhe, Germany |
| PBS |
| Gibco, Karlsruhe, Germany |
| Penicillin-Streptomycin solution |
| Gibco, Karlsruhe, Germany |
| Retinoic acid |
| Sigma, Steinheim, Germany |
| Retronectin |
| Takara, Shiga, Japan |
| RPMI 1640 |
| Gibco, Karlsruhe, Germany |
| Trypan blue stain (0.4%) |
| Gibco, Karlsruhe, Germany |
| Trypsin EDTA, 0.25% solution |
| Gibco, Karlsruhe, Germany |
| rmG-CSF |
| Peprotech, Offenbach, Germany |
| rmSCF |
| Peprotech, Offenbach, Germany |
| rmIL-6 |
| Peprotech, Offenbach, Germany |
| rmIL-3 |
| Peprotech, Offenbach, Germany |
| rmGM-CSF |
| Peprotech, Offenbach, Germany |
| Calf intestinal phosphatase (CIP) |
| NEB, Frankfurt, Germany |
| Gateway LR clonase enzyme mix |
| Invitrogen, Karslruhe, Germany |
| Klenow-Fragment DNA-polymerase I |
| NEB, Frankfurt, Germany |
| Restriction endonucleases |
| NEB, Frankfurt, Germany |
| RNAse |
| Sigma, Steinheim, Germany |
| Superscript II |
| Invitrogen, Karlsruhe, Germany |
| T4 DNA-ligase |
| NEB, Frankfurt, Germany |
| Taq-DNA-polymerase |
| Biosystems, Weiterstadt, Germany |
| Proteinase K |
| Stratagene, La Jolla, USA |
| Taq-DNA-polymerase |
| Biosystems, Weiterstadt, Germany |
| DNTPs |
| Fermentas, St. Leon-Rot, Germany |
| PCR Buffer |
| Fermentas, St. Leon-Rot, Germany |
| MgCl2 |
| Fermentas, St. Leon-Rot, Germany |
| PfU turbo polymerase |
| Stratagene, La Jolla, USA |
| Primers |
| Sigma, Steinheim, Germany |
| Mouse anti-β-tubulin (Ab-4) |
| Neo Markers, Asbach, Germany |
| Mouse lineage panel |
| Pharmingen, San Diego, CA, USA |
| Rabbit anti-c-ABL (11/24) |
| Santa Cruz, Heidelberg, Germany |
| Rabbit anti-p-ABL-Tyr-245 |
| Upstate-Biotechnology, Lake Placid, NY, |
| USA |
| Rabbit anti-p-ABL-Tyr-412 |
| Cell Signalling, Boston, USA |
| Rabbit anti-p-BCR-Tyr-177 |
| Cell Signalling, Boston, USA |
| Rabbit anti-BCR(C-20) |
| Santa Cruz, Heidelberg, Germany |
| Mouse anti- GFP |
| Upstate-Biotechnology, Lake Placid, NY, |
| USA |
| Mouse anti-phospho-STAT5 |
| Cell Signalling, Boston, USA |
| Rabbit anti -STAT5 |
| Cell Signalling, Boston, USA |
| Rabbit anti-phospho-CrkL (Tyr207) |
| Cell Signalling, Boston, USA |
| Mouse anti-CrkL |
| Cell Signalling, Boston, USA |
| Mouse anti-phosphotyrosine (clone 4G10) |
| Upstate-Biotechnology, Lake Placid, NY, |
| USA |
| Anti-mouse IgG-HRP |
| Santa Cruz, Heidelberg, Germany |
| Anti-rabbit IgG-HRP |
| Santa Cruz, Heidelberg, Germany |
| Mouse-anti-human-PE-LNGFR |
| BD, San Jose, CA, USA |
| Rat-anti- mouse-PE-Gr-1 |
| BD, San Jose, CA, USA |
| Rat-anti- mouse-PE-Mac-1 |
| BD, San Jose, CA, USA |
| Rat-anti- mouse-PE-B220 |
| BD, San Jose, CA, USA |
| Mouse-anti-PE-IgG 2a, K |
| BD, San Jose, CA, USA |
| Mouse-anti-PE-IgG 2b, K |
| BD, San Jose, CA, USA |
| Rat –anti-Mouse FITC-Sca-1 |
| BD, San Jose, CA, USA |
| Rat-anti- mouse-PE-Sca-1 |
| BD, San Jose, CA, USA |
| MACS buffer |
| 20% (w/v) BSA |
| 12.5 ml |
| EDTA |
| 1mmol |
| Penicillin/Streptomycin |
| 5ml |
| Add PBS up to |
| 500ml |
| 2M CaCl2 solution |
| The component was dissolved in ddH2O and sterile filtered with 0.22 μm filter and stored at – |
| 20°C in aliquots until use. |
| 2X HBS solution |
| Na2HPO4 |
| 0.315g (1.5 mM final) |
| HEPES |
| 19.5g (0.05M final) |
| NaC |
| 24g (0.28M final) |
| Water added up to 1200ml, three clean bottles were filled with exactly 400 ml of the solution, |
| the pH to 6,95/ 7,0 / 7,05 was adjusted respectively, each bottle was filled to 500 ml, pH was |
| controlled again. The buffer was filter-sterilized and stored at -20°C in aliquots. |
| 1x TAE Electrophoresis buffer |
| Tris-Acetate |
| 0,04M |
| EDTA |
| 1 mM |
| Buffer W for lysis of bacteria |
| Tris-HCl pH 8.0 |
| 100 mM |
| NaCl |
| 150 mM |
| EDTA |
| 1 mM |
| Tween |
| 0.1% |
| Protease inhibitor |
| 1x |
| 1X SDS gel-loading buffer |
| Tris-HCl, pH 6.8 |
| 50mM |
| SDS |
| 2 % (w/v) |
| Glycerol |
| 10% (v/v) |
| β-mercaptoethanol |
| 100mM |
| Bromophenol blue |
| 0.1 %( v/v) |
| 1X SDS gel-loading buffer lacking dithiothretitol was stored at room temperature. β- |
| mercaptoethanol from a 14 M stock was added just before the buffer is used. |
| For protein electrophoresis |
| (Invitrogen life technologies, Germany) |
| NuPAGE Novex Bis-Tris |
| Tris-Acetate Pre-Cast Gels |
| NuPAGE Novex MES and Tris-Acetate SDS Running Buffers |
| NuPAGE Transfer Buffer |
| Precision plus protein (PPP) |
| NuPAGE LSD Sample Buffer |
| NuPAGE Reducing Agent |
| NuPAGE Antioxidant |
| 10X TBS |
| Trizma HCl, pH 7.5 |
| volume was adjusted to 1 L with wate. |
| TBST |
| 1X TBS+ 0.05% Tween 20 |
| Blocking Buffer for immunoblot |
| 5% Carnation non-fat dry milk in TBST |
| Protein transfer buffer |
| TrisHCl |
| 48 mM |
| Glycine |
| Coomassie staining solution |
| Coomassie brilliant blue R250 |
| 0.1 % (w/v) |
| Methanol |
| 50 % (v/v) |
| Glacial acetic acid |
| 10 % (v/v) |
| Coomassie destaining Solution |
| Methanol |
| 50 % (v/v) |
| Glacial acetic acid |
| 10 % (v/v) |
| Ponceau S staining solution |
| Ponceau S |
| 2 % (w/v) |
| Trichloroacetic acid |
| 30 % (v/v) |
| Sulfosalicylic acid. |
| 30 % (v/v) |
| ECLsolution I: |
| 1 M TrisHCl, pH 8,5 |
| 10ml |
| 90mM p-Coumaric acid (in DMSO) 0.44ml |
| 250mM Luminol (in DMSO) 1ml |
| Water was dded up to 100ml; aliquots were made and stored in the dark at -20°C. |
| ECLsolution II: |
| 30 % H2O2 |
| 64μl |
| 1 M TrisHCl, pH 8,5 |
| 10 ml |
| Water was added up to 100ml, aliquat solution and store in the dark at -20°C. |
| Stripping buffer |
| Tris-HCl, pH8.0 |
| 62.5 mM |
| 2-mercaproethanol |
| 100 mM |
| SDS |
| 2 % (w/v) |
| FACS Wash Buffer |
| PBS + 1%FCS + 1% NaN3 |
| FACS Fixative Solution |
| PBS + 2% formaldehyde solution |
| Phosphate-Buffered Saline (PBS) |
| NaCl |
| 137 mM |
| KCl |
| 2.7 mM |
| Na2HPO4 |
| 10 mM |
| KH2PO4 |
| 2 mM |
| The above components were dissoolved in water and adjusted pH to 7.4 with HCl. |
| Tris-HCl (1M) |
| 121.1g of Tris base was dissolved in 800ml of water. The pH was adjusted to the desired |
| 10X Tris EDTA (TE) |
| pH 7.4 |
| 100 mM Tris-Cl (pH 7.4) |
| 10 mM EDTA (pH 8.0) |
| pH 7.6 |
| 100 mM Tris-Cl (pH 7.6) |
| 10 mM EDTA (pH 8.0) |
| pH 8.0 |
| 100 mM Tris-Cl (pH 8.0) |
| 10 mM EDTA (pH 8.0) |
| SDS (20% w/v) |
| 200 g of electrophoresis-grade SDS was dissolved in 900 ml of water. The mixture was |
| heated to 68°C and stirred with a magnetic stirrer to assist dissolution. The pH was adjusted to |
| 7.2 by adding a few drops of concentrated HCl. The volume was adjusted to 1 liter with |
| water. |
| EDTA (0.5 M, pH 8.0) |
| 186.1 g of disodium EDTA was added to 800 ml of H2O. The mixture was stired vigorously |
| on a magnetic stirrer. The pH was adjusted to 8.0 with NaOH. The disodium salt of EDTA did |
| not go into solution until the pH of the solution was adjusted to ～ 8.0 by the addition of |
| NaOH. |
| Page 49 |
| Materials |
| 49 |
| Solutions for plasmid minipreparation |
| Resuspension solution (Sol I) |
| Glucose |
| 50 mM |
| Tris-HCl pH 8.0 |
| 25 mM |
| EDTA pH 8.0 |
| 10 mM |
| Autoclaved and stored the solution at 4°C |
| Alkaline lysis solution (Sol II) |
| NaOH |
| 0.2 M |
| SDS |
| 1 % (w/v) |
| Sol II was prepared fresh and used at room temperature |
| Neutralization solution (Sol III) |
| 5 M Potassium acetate |
| 60 ml |
| Glacial acetic acid |
| 11.5 ml |
| ddH2O 28.5 ml |
| The solution was stored at 4°C and transferred to ice before use |
| 0.1% Diethylpyrocarbonate (DEPC) |
| 1 g DEPC was dissolved in 1 L water and mixed it vigiously. It let stand with loose lid |
| overnight at room temperature or at 37°C for 1h and autoclaved for 15 minutes at 15 psi on |
| liquid cycle. DEPC cannot be used to treat Tris buffer. |
| 2.3.7 Plasmids and vectors |
| pCDNA 3.1 vector |
| Invitrogen GmbH, Karlsruhe, Germany |
| pEntr1A |
| Invitrogen GmbH, Karlsruhe, Germany |
| PINCO |
| Retroviral hybrid vector with LTR derived from Moloney |
| murine Leukemia virus. The main characteristics of this vector |
| is the presence of the EBV origin of replication and the EBNA- |
| 1 gene and the presence of the cDNAs that encodes for the |
| EGFP, controlled by a cytomegalovirus promoter.This vector |
| allows high-efficiency gene transfer (Grignani et al., 1998). |
| PAULO |
| This vector is a modified pinco vector in which GFP is replaced |
| by LNGFR |
| PIDE |
| This vector is based on pinco vector in which GFP is deleted and |
| Hind III site can be used for cloning gene instead of GFP |
| Pinco∆CMV |
| Retroviral pinco based vector without any reporter gene |
| Page 50 |
| Materials |
| 50 |
| pPRIBA2 |
| IBA, Gottingen , Germany |
| pGEX4T3 |
| Amersham Biosciences, Freiburg, Germany |
| 2.3.8 Bacterial E.Coli Strain and genotype |
| E. coli – HB101, BL21, JM83, DB 3.1, |
| Invitrogen, Karlsruhe, Germany |
| 2.3.9 Medium for bacterium |
| LB medium |
| Bacto-Tryptone |
| 1 % (w/v) |
| Bacto-Yeast-Extrac |
| 0.5 % (w/v) |
| NaCl |
| 1.5 % (w/v) |
| Adjuted pH to 7.4 with NaOH and autoclaved |
| LB agar plates |
| Bacto-Agar |
| 1.5 % (w/v) |
| Bacterium freezing solution |
| Glycerin |
| 65 % (v/v) |
| MgSO4 |
| 0.1 M |
| TrisHCl, pH 8.0 |
| 0.025M |
| SOC |
| Invitrogen, Karlsruhe, Germany |
| 2.3.10 Cell lines |
| 2.3.10.1 Ph+ cells |
| CML blast cell lines |
| In CML cells, the translocation product encodes for p210 |
| BCR/ABL |
| . |
| BV173: Human B cell precursor leukaemia cell line, established from the peripheral blood of |
| a patient with chronic myeloid leukaemia (CML) in lymphoid blast crisis. Contains the t(9;22) |
| b2-a2 fusion gene. Obtained from DSMZ, Germany |
| K562: Established from the pleural effusion of a patient with chronic myeloid leukaemia |
| (CML) in myeloid blast crisis. Cells carry the Ph chromosome with the Bcr-Abl b3-a2 fusion |
| gene. Obtained from DSMZ, Germany |
| Ph+ ALL |
| In Ph+ cells, the translocation product encodes for p185 |
| BCR/ABL |
| . |
| Page 51 |
| Materials |
| 51 |
| Sup-B15: Human B cell precursor leukemia cell line, established from the bone marrow of a |
| patient with acute lymphoblastic leukemia, carrying the ALL-variant (m-bcr) of the |
| BCR/ABL fusion gene (e1-a2). Obtained from DSMZ, Germany |
| Tom-1: human B cell precursor leukemia. Established from the bone marrow of a patient with |
| refractory Ph chromosome acute lymphoblastic leukemia (ALL); described to carry the ALL- |
| variant (m-BCR) of the BCR/ABL fusion gene (e1-a2) obtained from DSMZ, Germany. |
| 2.3.10.2 Other Cell lines |
| Nalm-6: Human lymphoblastic B cell line; Ph- |
| Ba/F3: IL-3 dependent murine pro B cell line established from peripheral blood; apparently |
| derived from BALB/c mouse. |
| 32D: IL-3 dependent murine myeloid cell line established from peripheral blood. |
| Rat-1: Rat fibroblast cells line |
| 293T: Transformed human embryonal kidney cell line |
| Phoenix: Based on the 293T cell line (transformed human embryonal kidney cell line). |
| Expresses the retroviral structural genes gag, pol und env. It is also known as a packaging cell |
| line. The ecotropic packaging cell line delivers genes only to dividing murine or rat cells. |
| All cells were obtained from the “Deutsche Sammlung von Mikroorganismen und |
| Zellkulturen GmbH” (DSMZ) (German Collection of Microorganisms and Cell Cultures), |
| with the exception of the ecotrophic Phoenix cells which were obtained from Nolan lab, |
| Standford, USA. |
| 2.3.11 Medium for Cell culture |
| L-glutamine |
| 1 % (v/v) |
| FBS |
| 10 % (v/v) |
| Penicillin/Streptomycin |
| 1 % (v/v) |
| Added to DMEM and RPMI medium for adherent and suspension cells respectively |
| Medium for 32D/Baf3 cells |
| L-glutamine |
| 1 % (v/v) |
| FBS |
| 10 % (v/v) |
| Penicillin/Streptomycin |
| 1 % (v/v) |
| mIL-3 |
| 10 ng /ml |
| Added the above to RPMI medium |
| Medium for Nalm-6, BV-173 and K562 |
| L-glutamine |
| 1 % (v/v) |
| Page 52 |
| Materials |
| 52 |
| FBS |
| 10 % (v/v) |
| Penicillin/Streptomycin |
| 1 % (v/v |
| Added the above to RPMI medium |
| Medium for Tom-1 |
| L-glutamine |
| 1 % (v/v) |
| FBS |
| 20 % (v/v) |
| Penicillin/Streptomycin |
| 1 % (v/v |
| Added the above to RPMI medium |
| Medium for Sup-B15 |
| L-glutamine |
| 1 % (v/v) |
| FBS |
| 20 % (v/v) |
| Penicillin/Streptomycin |
| 1 % (v/v |
| Added the above to Mycosa medium |
| Medium for Sca1+/lin- mouse BM cells |
| L-glutamine |
| 1 % (v/v) |
| FBS(Hyclon) |
| 10 % (v/v) |
| Penicillin/Streptomycin |
| 1 % (v/v) |
| mIL-3 |
| 20 ng /ml |
| mIL-6 |
| 20 ng /ml |
| mSCF |
| 100 ng /ml |
| Added the above to ISCOVE medium |
| Cell freezing medium |
| Solution I |
| RPMI/DMEM |
| 70 % (v/v) |
| FBS |
| 30 % (v/v) |
| Solution II |
| RPMI/DMEM |
| 80 % (v/v) |
| DMSO |
| 20 % (v/v) |
| 2.3.12 Materials for animal experiments |
| 2.3.12.1 Mice |
| The C57BL/6N mice (age between 6-8 weeks, all female) were purchased from Charles River |
| Laboratories GmbH in Munich, Germany and served as the recipient mice for all the animal |
| experiments. |
| Page 53 |
| Materials |
| 53 |
| 2.4 Miscellaneous |
| Nucleospin |
| R |
| Mini plasmid DNA isolation Kit |
| Macherey-Nagel, Dueren Germany |
| Nucleospin |
| R |
| Maxi plasmid isolation kit |
| Macherey-Nagel, Dueren Germany |
| Gel cleaning kit |
| Macherey-Nagel, Dueren Germany |
| PCR cleaning kit |
| Macherey-Nagel, Dueren Germany |
| Qiagen gel extraction Kit |
| Qiagen, Duesseldorf, Germany |
| Qiagen Plasmid kit Mini, Midi and Maxi |
| Qiagen, Duesseldorf, Germany |
| Qiagen PCR purification Kit |
| Qiagen, Duesseldorf, Germany |
| Quick change site mutagenesis kit |
| Stratagene, La Jolla, USA |
| Endo Toxin removal kit |
| Profos AG, Regensburg, Germany |
| Cell proliferation (XTT) kit |
| Amersham/Pharmacia Biotech, Freiburg, |
| Germany |
| Protein concentrator (Vivapore) |
| VivaScience, Hannover, Germany |
| Page 54 |
| Methods |
| 54 |
| 3 Methods |
| 3.1 Preparation of plasmid DNA |
| 3.1.1 Transformation of E.coli |
| A frozen vial of competent E.coli bacteria was thawed on ice. The transforming DNA (up to |
| 25ng per 50ul competent bacterium or 10ul ligation product) was pipetted directly to |
| competent E.coli bacteria and mixed by swirling the tubes gently several time. |