To generate AML on an Erg-deficient background, c-Kit-enriched BM from Ergfl/fl and Ergfl/fl; R26-CreER mice were transduced with MLL-ENL-IRES-GFP (Ohlsson et al. 2014 (link)) and transformed through three passages in colony assays (Stem Cell Technologies, M3231) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 20 ng/mL murine SCF (mSCF) (PeproTech), 10 ng/mL human IL-6 (hIL-6) (PeproTech), 10 ng/mL human GM-CSF, and 10 ng/mL murine IL-3 (PeproTech). Ergfl/fl and Ergfl/fl; R26-CreER transformed colonies were subsequently dissociated and passaged in RPMI 1640 (Invitrogen) supplemented with 20% FCS, 20% WEHI-conditioned medium, 20 ng/mL mSCF, and 10 ng/mL hIL-6 ± 0.5 μM 4-OHT to allow for excision of Erg. Finally, serial replating assays were performed as described in Ohlsson et al. (2014) (link). For T-ALL analysis, c-Kit-enriched BM from Ergfl/fl and Ergfl/fl; Cd2iCre mice was transduced with NOTCH-ICD (Chiang et al. 2008 (link)) or NrasG12D in which luciferase was replaced with Venus (Zuber et al. 2009 (link)) and transplanted to irradiated (900 cGy) Ly5.1 recipients (CD45.1). Log rank test was used to analyze differences in survival.
Human il 6 hil 6
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Human IL-6 (hIL-6) is a recombinant protein that corresponds to the interleukin-6 (IL-6) cytokine found in humans. IL-6 is a pleiotropic cytokine involved in various biological processes, including the regulation of the immune response, hematopoiesis, and the acute phase response.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Human gm csf, by Thermo Fisher Scientific (1 mentions)
Murine il 3, by Thermo Fisher Scientific (1 mentions)
M3231, by STEMCELL (1 mentions)
Hil 6, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Sb202190, by Merck Group (1 mentions)
U0126, by Merck Group (1 mentions)
Alkaline phosphatase conjugated streptavidin, by Vector Laboratories (1 mentions)
Cd34 microbead kit ultrapure, by Miltenyi Biotec (1 mentions)
Evans blue, by Fujifilm (1 mentions)
Biotin conjugated anti mouse igg, by Agilent Technologies (1 mentions)
3 protocols using human il 6 hil 6
1
AML Generation in Erg-deficient Model
2
Cytokine Signaling in Cell Lines
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Human dermal fibroblasts (HDF), lung fibroblasts (HLF) and osteosarcoma cells (U2OS) were grown in DMEM. Human hepatoma cells (HepG2) were grown in DMEM‐F12. All media were purchased from Invitrogen (Darmstadt, Germany) and supplemented with 10% FCS (PAA, Cölbe, Germany). All cells were cultured at 37°C under 5% CO2 in a water‐saturated atmosphere. Recombinant human OSM (hOSM), human IFN‐γ (hIFN‐γ) and human IL‐6 (hIL‐6) were purchased from Peprotech (Hamburg, Germany) and recombinant human LIF from Sigma‐Aldrich (Taufkirchen, Germany). Soluble human IL‐6Rα (sIL‐6R) was generated as described 34 and kindly provided by Prof. Dr. G. Müller‐Newen (RWTH Aachen University Hospital). LPS was obtained from Cell Concepts (Umkirch, Germany). U0126 and SB202190 were obtained from Merck Millipore (Darmstadt, Germany). The double‐stranded polyribonucleotide poly(I)·poly(C) (poly I:C) was obtained from GE Healthcare (Amersham, United Kingdom).
3
Megakaryocyte Colony Formation Assay
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A Mk colony formation assay was performed as described previously33 (link),34 (link) with minor modifications. CD34-positive cells were purified from cryopreserved bone marrow mononuclear cells (BM-MNCs) using CD34 MicroBead Kit UltraPure (MACS Miltenyi Biotec). CD34-positive cells (5000) were cultured in a chamber (two-chamber slide, Matsunami) with serum-free collagen medium in the presence of 20 ng/mL human IL-3 (hIL-3) (Miltenyi Biotec) and human IL-6 (hIL-6) (Peprotech) and in the presence or absence of 50 ng/mL human thrombopoietin (hTPO) (Kyowa Kirin). The cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air for 12–14 days. Fixed cells were stained with anti-CD41 antibody (Clone HIP8, STEMCELL), biotin-conjugated anti-mouse IgG (Dako), streptavidin-conjugated alkaline phosphatase (Vector Laboratory), SIGMA FAST Red TR/naphthol AS-MX alkaline phosphatase substrate (Sigma), and Evans Blue (FUJIFILM Wako). BM-MNCs collected from lymphoma patients without BM infiltration and reactive thrombocytosis patients were used as negative controls.
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