Hil 6

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The HIL-6 is a lab equipment product designed for general laboratory use. It serves as a high-intensity light source, providing illumination for various applications within the laboratory setting. The core function of the HIL-6 is to generate and deliver consistent, high-intensity light to enable various experimental and analytical procedures.

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Human IL-6 (hIL-6) (3 citations)

30 protocols using hil 6

Expansion of Transduced CD34+ Cells

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Transduced CD34+ cells were co-cultured with irradiated (80 Gy) murine fibroblasts that constitutively secrete human growth factors (M2-10B4, CLS Cell Lines Service). Before irradiation, M2-10B4 cells were grown in RPMI medium (Biochrom) with 10% FCS (FBS Superior, Biochrom) and 1% Penicillin-Streptomycin (Biochrom). CD34+ cells were cultured in Stem Span medium supplied with cytokines (human Flt3-Ligand, hFlt3 100 ng/ml, human stem cell Factor, hSCF 100 ng/ml, human thrombopoietin, hTPO 20 ng/ml, human interleukin 6, hIL-6 20 ng/ml, Peprotech) and 1% Penicillin-Streptomycin (Biochrom), through the transduction and sorting of transduced cells. Transduced CD34+ cells were grown for at least 6 weeks on irradiated M2-10B4 cells according to standard procedures in MyeloCult (Stem Cell Technologies). For optimal growth hydrocor-tisone (10⁻⁶ M, Stem Cell Technologies) and cytokines (hFlt3 20 ng/ml, hSCF 20 ng/ml, hTPO 4 ng/ml, hIL-6 4 ng/ml, Peprotech) were added. On a weekly basis, half the medium was changed and used for analysis.

Salari A., Thomay K., Lentes J., Ebersold J., Hagedorn M., Skawran B., Davenport C., Schambach A., Schlegelberger B, & Göhring G. (2018). Effect of TP53 contact and conformational mutations on cell survival and erythropoiesis of human hematopoietic stem cells in a long term culture model. Oncotarget, 9(52), 29869-29876.

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Lentiviral ShRNA Knockdown in Leukemia Cells

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Npm1c/Flt3-ITD leukemia cells (termed DM cells) were derived by isolation of Lin- BM HSPC from double mutant mice and were cultured in X-Vivo medium (Lonza) plus 10% fetal bovine serum (Gibco) in the presence of 10 ng/mL mIL-3, 10 ng/mL hIL-6, and 50 ng/mL mSCF (Peprotech). Lentiviral shRNA plasmids were obtained from Sigma-Aldrich in the form of MISSION pLKO.1-puro vectors and shRNA sequences were provided in Supplementary Table 12. Lentiviral particles were produced by co-transfection of shRNA plasmids with psPAX and pMDG.2 in 293T cells using the Trans-IT LT-1 transfection reagent (Mirus). 293T cells were cultured in DMEM (Gibco) plus 10% fetal bovine serum (Gibco). DM cells were infected by shRNA lentivirus twice via spinoculation in the presence of 5 μg/mL polybrene (Sigma-Aldrich). 72 hours post transduction, cells were selected with 2 μg/mL puromycin (Sigma-Aldrich) for 72 hours.

Yun H., Narayan N., Vohra S., Giotopoulos G., Mupo A., Madrigal P., Sasca D., Lara-Astiaso D., Horton S.J., Agrawal-Singh S., Meduri E., Basheer F., Marando L., Gozdecka M., Dovey O.M., Castillo-Venzor A., Wang X., Gallipoli P., Müller-Tidow C., Osborne C.S., Vassiliou G.S, & Huntly B.J. (2021). Mutational synergy during leukemia induction remodels chromatin accessibility, modification and 3-Dimensional DNA topology to alter gene expression. Nature genetics, 53(10), 1443-1455.

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Expansion of Primary NK Cells from Peripheral Blood and Cord Blood Hematopoietic Stem Cells

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Peripheral blood mononuclear cells (PBMCs), procured from the peripheral blood of healthy donors, were segregated using density gradient centrifugation facilitated by Ficoll-Hypaque (1.077 g/mL). To facilitate PB-NK cell expansion, thawed PBMCs were combined with 2 × 10⁵ K562-mbIL21-feeder cells at a proportion of 1 × 10⁵ cells per ml in StemSpan™ SFEM II medium (Stemcell Technologies). The medium was enriched with 1% L-glutamine (Invitrogen), 100 ng/ml hIL-2 (Peprotech), 20 ng/ml hIL-7 (Peprotech), 20 ng/ml hIL-15 (Peprotech), and 50 μg/ml ascorbic acid (Sigma) [20 (link)]. Both the cytokines and ascorbic acid were freshly added prior to use.
Cord blood CD34⁺ HSPCs were isolated using the CD34 MicroBead Kit (Miltenyi Biotec) [21 (link)]. The enriched HSPC population comprised over 90% CD34⁺ cells. These HSPCs were introduced at 5 × 10⁵ cells per ml into serum-free StemSpan™ SFEM II medium (Stemcell Technologies). The medium was enriched with 1% L-glutamine (Invitrogen), 100 ng/ml hSCF (Peprotech), 100 ng/ml hFlt3-L (Peprotech), 100 ng/ml hTPO (Peprotech), 50 ng/ml hIL-6 (Peprotech), 750 nM SR1 (Sigma), and 50 nM UM171 (Sigma). To maintain optimal cell density, fresh medium was administered every two days before initiating NK cell differentiation, thus ensuring a cell density range of 5 × 10⁵ to 1 × 10⁶ cells per ml.

Wen W., Chen X., Shen X.Y., Li H.Y., Zhang F., Fang F.Q, & Zhang X.B. (2023). Enhancing cord blood stem cell-derived NK cell growth and differentiation through hyperosmosis. Stem Cell Research & Therapy, 14, 295.

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Transduction of Patient-Derived AML Cells

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Patient-derived AML samples were obtained from The Ohio State Comprehensive Cancer Center and used according to the approved IRB protocol 16-9037. Cells were plated on irradiated mono-layers of HS27 cells (21 (link)) and cultured in Stemspan (Stem Cell Technology) supplemented with 10% FBS, 100ng/ml hSCF (Peprotech), 100ng/ml hFLT3 ligand (Peprotech), 20ng/ml hIL-3 (Peprotech), 20ng/ml hIL-6 (Peprotech), 20ng/ml G-CSF (Peprotech). 500,000 cells were transduced with pLKO.1 GFP lentiviral shRNA vectors and evaluated for GFP expression every 3 days for 12 days after staining with human CD45 APC-Cy7 (BD Biosciences) and Propidium Iodide (PI) by flow cytometry.

Di Marcantonio D., Martinez E., Sidoli S., Vadaketh J., Nieborowska-Skorska M., Gupta A., Meadows J.M., Ferraro F., Masselli E., Challen G.A., Milsom M.D., Scholl C., Fröhling S., Balachandran S., Skorski T., Garcia B.A., Mirandola P., Gobbi G., Garzon R., Vitale M, & Sykes S.M. (2017). PKC epsilon is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(3), 608-618.

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Retroviral Transduction of Mouse Bone Marrow

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Retroviral vectors MSCV-HoxA9-PGKneo, pSF91-IRES-eGFP, pSF91-IDH1mut-IRES-eGFP, pSF91-IDH1wt-IRES-eGFP have been described previously, (19 (link)) and MSCV-MLL-AF9-IRES-eGFP was a kind gift from Dr. Florian Kuchenbauer. Primary mouse bone marrow cells from C57BL/6J mice were transduced with helper-free recombinant retrovirus as previously described.(19 (link)) In brief, bone marrow cells were harvested from 5-FU (Medac, Hamburg, Germany) treated mice, and were infected first by cocultivation with a HoxA9 viral producer cell line followed by either control (CTL) vector or IDH1wt or IDH1mut viral producer GP+E86 cells. Alternatively, prestimulated bone marrow cells were transduced with MLL-AF9. Cells were then sorted for GFP expression and maintained in 6 ng/ml mIL-3, 10 ng/ml hIL-6, and 20 ng/ml mSCF (Peprotech, Hamburg, Germany).

Chaturvedi A., Araujo Cruz M.M., Jyotsana N., Sharma A., Goparaju R., Schwarzer A., Görlich K., Schottmann R., Struys E.A., Jansen E.E., Rohde C., Müller-Tidow C., Geffers R., Göhring G., Ganser A., Thol F, & Heuser M. (2016). Enantiomer-specific and paracrine leukemogenicity of mutant IDH metabolite 2-hydroxyglutarate. Leukemia, 30(8), 1708-1715.

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Lentiviral Transduction of HSPCs

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Lentivirus was produced and concentrated as previously described^{4 (link)}. A high-titre virus of IK6-GFP and Ctrl-GFP was prepared by ultracentrifugation. Lin⁻CD34⁺CD38⁻ HSPCs were infected with lentivirus at a multiplicity of infection of 100 for 24 h in serum-free Stem/Span (Stem Cell Technologies, Canada) medium supplemented with growth factors (hSCF 50 ng/ml, hFlt3-L 50 ng/ml, hIL-6 10 ng/ml and hTPO 10 ng/ml; Peprotech, USA).

Li Z., Li S.P., Li R.Y., Zhu H., Liu X., Guo X.L., Mu L.L., Cai J.J., Bai F., Chen G.Q, & Hong D.L. (2018). Leukaemic alterations of IKZF1 prime stemness and malignancy programs in human lymphocytes. Cell Death & Disease, 9(5), 526.

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Establishment of Mouse Leukemic Cell Lines

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Establishment of mouse leukemic cell lines were done by culturing sorted GFP⁺mCherry⁺ (for KMT2A-MLLT3 + FLT3^N676K, KMT2A-MLLT3 + FLT3^N676K and KMT2A-MLLT3 + NRAS^G12D) or mCherry⁺ (for KMT2A-MLLT3 + Empty-GFP) leukemic BM cells for 1 week in “C10” media, consisting of RPMI-1640 with l-glutamine (Thermo Scientific), 10% fetal bovine serum (FBS, Thermo Scientific), 100 units/mL Penicillin and 100 g/ml Streptomycin (Thermo Scientific), 55 µM ß-mercaptoethanol (Sigma-Aldrich), 0.1 mM non-essential amino acids (Sigma-Aldrich), 1 mM sodium pyruvate (Thermo Scientific) and 10 mM HEPES (Thermo Scientific) supplemented with 100 ng/ml mSCF, 10 ng/ml hIL6 and 10 ng/ml mIL3 (Peprotech)^{10 (link)}, after which mSCF and hIL6 were withdrawn and mIL3 was decreased to 1 ng/ml for continuous culturing.

Hyrenius-Wittsten A., Pilheden M., Sturesson H., Hansson J., Walsh M.P., Song G., Kazi J.U., Liu J., Ramakrishan R., Garcia-Ruiz C., Nance S., Gupta P., Zhang J., Rönnstrand L., Hultquist A., Downing J.R., Lindkvist-Petersson K., Paulsson K., Järås M., Gruber T.A., Ma J, & Hagström-Andersson A.K. (2018). De novo activating mutations drive clonal evolution and enhance clonal fitness in KMT2A-rearranged leukemia. Nature Communications, 9, 1770.

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Cultured Cell Lines and Patient-Derived AML Cells

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HoxA9-IDH1R132H, HoxA9-IDH1R132C, HoxA9-IDH2R140Q and HoxA9-IDH2172K cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 10 ng/mL of human interleukin 6 (hIL-6), 6 ng/mL of murine interleukin 3 (mIL-3), and 20 ng/mL of murine stem cell factor (mSCF; all from Peprotech) and were incubated at 37°C with 5% CO₂ in humidified atmosphere. Patient-derived AML cells, freshly isolated or cryopreserved, were cultured in IMDM medium (Gibco) supplemented with 12,5% FBS (Gibco), 12,5% horse serum (Gibco), 5 µM hydrocortisone, 2,5 mM GlutaMax, 10 ng/ml human FLT3L, 10 ng/ml human TPO, 50 ng/µl human SCF and 10 ng/µl human IL-3 (all from Peprotech) and were incubated at 37°C with 5% CO₂ in humidified atmosphere. Treatment was carried out with BAY1436032 or DMSO for indicated time-points and concentrations.

Chaturvedi A., Herbst L., Pusch S., Klett L., Goparaju R., Stichel D., Kaulfuss S., Panknin O., Zimmermann K., Toschi L., Neuhaus R., Haegebarth A., Rehwinkel H., Hess-Stumpp H., Bauser M., Bochtler T., Struys E.A., Sharma A., Bakkali A., Geffers R., Araujo-Cruz M.M., Thol F., Gabdoulline R., Ganser A., Ho A.D., von Deimling A., Rippe K., Heuser M, & Krämer A. (2017). Pan-mutant-IDH1 inhibitor BAY1436032 is highly effective against human IDH1 mutant acute myeloid leukemia in vivo. Leukemia, 31(10), 2020-2028.

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CD34+ Cells Transduction Protocol

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CD34+ cells were cultured in Stem Span medium (Stem Cell Technologies) containing cytokines (hFlt3 100 ng/ml, hSCF 100 ng/ml, hTPO 20 ng/ml, hIL-6 20 ng/ml, Peprotech) and 1% Penicillin-Streptomycin (Biochrom) 12 h before transduction. Transduction of pre-stimulated cells was done in RetroNectin (Takara, Shiga, Japan)-coated plates according to the manufacturer's instructions. Briefly, cells were seeded in a coated 6-well plate, then concentrated viral supernatants (MOI 2.5) and protamine sulfate (4 μg/ml) were added to the cells and the plate was centrifuged for 2 h at 31°C, 1,500 × g. Cells were incubated for 12 h, then fresh medium was added to the cells and 12 h later medium was exchanged. 48 h after transduction cells were sorted for GFP⁺ cells by fluorescence-activated cell sorting (FACS).

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Culturing Leukemia Cell Lines and Patient-Derived AML Cells

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HoxA9-IDH1R132H, HoxA9-IDH1R132C, HoxA9-IDH2R140Q and HoxA9-IDH2172K cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum (FBS), 10 ng/ml of human interleukin 6 (hIL-6), 6 ng/ml of murine interleukin 3 (mIL-3), and 20 ng/ml of murine stem cell factor (mSCF; all from PeproTech, Hamburg, Germany) and were incubated at 37 °C with 5% CO₂ in humidified atmosphere. Patient-derived AML cells, freshly isolated or cryopreserved, were cultured in IMDM medium (Gibco, Karlsruhe, Germany) supplemented with 12.5% FBS (Gibco), 12.5% horse serum (Gibco), 5 μm hydrocortisone, 2.5 mm GlutaMax, 10 ng/ml human FLT3-ligand, 10 ng/ml human TPO, 50 ng/μl human SCF and 10 ng/μl human IL-3 (all from PeproTech) and were incubated at 37 °C with 5% CO₂ in humidified atmosphere. Treatment was carried out with BAY1436032 or dimethyl sulfoxide for indicated time points and concentrations.

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