Iscove s modified dulbecco s medium

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Iscove's modified Dulbecco's medium is a cell culture medium formulation commonly used for the in vitro cultivation of various cell types, including hematopoietic cells and some types of mammalian cells. The medium provides a balanced salt solution and a variety of nutrients required for cell growth and maintenance.

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Iscove’s Modified Dulbecco Medium Iscove’s modified Dulbecco medium (IMDM; Complete Iscove's modified Dulbecco's medium Iscove’s Dulbecco’s modified Eagle’s medium Iscove's modified Dulbecco's Modified Eagle's Medium Iscove’s Modified Dulbecco’s Medium (IMDM) Gibco® Iscove's modified Dulbecco's medium (IMDM; Iscove modified Dulbecco medium Iscove’s medium Dulbecco’s modified

425 protocols using iscove s modified dulbecco s medium

Isolation and Culture of Murine and Human Cells

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Female C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd. (Beijing Laboratory Animal Research Center), and maintained in isolator cages under specific pathogen-free conditions. All mice were used at 6-8 week of age. The experimental manipulation of mice was undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Science & Technology of Jilin Province.
Human PBMCs were isolated from buffy coats (Blood Center of Jilin Province, China) by Ficoll_Hypaque density gradient centrifugation (Pharmacia) and washed three times with Iscove's modified Dulbecco's medium (GIBCO). The viability of the PBMCs was 95-99% as determined by trypan blue exclusion. Murine B16 melanoma cells of C57BL/6 origin and Vero E6 cells (African green monkey kidney cell line, American Type Culture Collection) were grown in Iscove's modified Dulbecco's medium (GIBCO) supplemented with 10% heat-inactivated FBS (GIBCO), 0.15% NaHCO₃, 100 IUmL^-1 penicillin, 100 IUmL^-1 streptomycin, and 2 mM L^-1glutamine at 37 °C in humidified air containing 5% CO₂31 (link).

Sun W., Fang M., Chen Y., Yang Z., Xiao Y., Wan M., Wang H., Yu Y, & Wang L. (2016). Delivery System of CpG Oligodeoxynucleotides through Eliciting an Effective T cell Immune Response against Melanoma in Mice. Journal of Cancer, 7(3), 241-250.

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Generation of Hematopoietic Stem Cells from iPSCs

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Patient-specific iPSCs were differentiated into hematopoietic stem cells (HSCs) under defined, serum-free and feeder-free conditions as previously reported.^{20 (link)} Briefly, TrypLE (Invitrogen)-dissociated iPSCs were seeded onto 6-well plates that were precoated with 3 mg cm⁻² human plasma fibronectin (Invitrogen) in mTeSR1 supplemented with an inhibitor of Rho-associated kinase (H1152, Sigma). After 1 day, mTeSR1 was replaced with a hematopoietic commitment medium containing Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 1 × HIT (Stem Cell Technologies), 450 μM monothioglycerol (Sigma), 50 ng ml⁻¹ recombinant human BMP4 (R&D Systems), 50 ng ml⁻¹ recombinant human vascular endothelial growth factor (Invitrogen), 0.1 mM NEAA (Invitrogen) and 2 mM L-glutamine (Invitrogen). After 6 days, the medium was changed to a hematopoietic maturation medium consisting of Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 5 U ml⁻¹ heparin (Sigma), 25 ng ml⁻¹ thrombopoietin (Invitrogen), 25 ng ml⁻¹ human recombinant stem cell factor (Invitrogen), 25 ng ml⁻¹ human recombinant FLT3L (Peprotech, Rocky Hill, NJ, USA), 10 ng ml⁻¹ interleukin-3 (Invitrogen) and 10 ng ml⁻¹ interleukin-6 (Invitrogen). The cells were incubated under hypoxic conditions (5% O₂ balanced with nitrogen).

Son M.Y., Lee M.O., Jeon H., Seol B., Kim J.H., Chang J.S, & Cho Y.S. (2016). Generation and characterization of integration-free induced pluripotent stem cells from patients with autoimmune disease. Experimental & Molecular Medicine, 48(5), e232-.

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Culturing Primary Cells and CML Cell Lines

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Primary cells were cultured in SFM supplemented with stem cell factor (SCF; 0.2 ng/ml; BioLegend, #573902), granulocyte colony-stimulating factor (G-CSF; 1 ng/ml; BioLegend, #578602), granulocyte-macrophage CSF (GM-CSF; 0.2 ng/ml; BioLegend, #572902), interleukin-6 (IL6; 1 ng/ml; BioLegend, #570802), macrophage inflammatory protein α (MIPα; 0.2 ng/ml; PeproTech, #300-08), leukemia inhibitory factor (LIF; 0.05 ng/ml; PeproTech, #300-05), [bovine serum albumin (BSA), insulin, and transferrin (BIT)] (20%; STEMCELL Technologies, #09500), low-density lipoprotein (40 μg/ml; Sigma-Aldrich, #L4646), 2-mercaptoethanol (0.1 mM; Invitrogen, #31350-010), and 1% penicillin/streptomycin (LifeTech, #15140-122) and resuspended in Iscove’s modified Dulbecco’s medium (LifeTech, #12440-053). The CML cell lines K562 and KCL22 (DSMZ) were cultured in RPMI 1640 medium (LifeTech, #11875-093) supplemented with 1% penicillin/streptomycin, 1% l-glutamine, and 10% (v/v) fetal calf serum. Both primary cells and cell lines were passaged every 2 to 3 days, maintained at a concentration of 2 × 10⁵ cells/ml at 37°C with 5% CO₂, and routinely tested for mycoplasma. ULK1 KO single-cell cloning was performed with serial dilution in a 96-well plate (100 μl per well). Puromycin selection (3 μg/ml) was performed after cell expansion, and Western blotting was performed to validate ULK1 KO.

Ianniciello A., Zarou M.M., Rattigan K.M., Scott M., Dawson A., Dunn K., Brabcova Z., Kalkman E.R., Nixon C., Michie A.M., Copland M., Vetrie D., Ambler M., Saxty B, & Helgason G.V. (2021). ULK1 inhibition promotes oxidative stress–induced differentiation and sensitizes leukemic stem cells to targeted therapy. Science translational medicine, 13(613), eabd5016.

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DLBCL Cell Culture and BCR Stimulation Assay

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The DLBCL cell lines TMD8, HBL-1, DB and OCI-Ly19 were grown in RPMI 1640 medium (HyClone Laboratories, Logan, UT) supplemented with glutamine, beta-mercaptoethanol, penicillin/streptomycin and 10% fetal bovine serum (FBS). Other DLBCL cell lines, OCI-Ly3 and OCI-Ly10, were maintained in Iscove’s modified Dulbecco’s medium (Life Technologies [Gibco], Grand Island, NY) supplemented with beta-mercaptoethanol, penicillin/streptomycin and 20% heparinized human plasma. All cell lines were grown in a humidified 5% CO₂ incubator at 37°C. Ibrutinib (PCI-32765), was provided by Pharmacyclics, Inc. (Sunnyvale, CA) and Idelalisib and P505-15 were purchased from Selleckchem (Houston, TX). The kinase inhibitors were stored as stock solutions of 10 mM in 100 % dimethyl sulfoxide at −20 °C. These stock solutions were diluted in complete RPMI medium with 10% FBS, L-glutamine (HyClone Laboratories) and penicillin-streptomycin (Cellgro, Hemdon, VA), and added to the assay medium to the indicated final concentrations. For BCR stimulation culture medium was supplemented with 10 μg/ml anti-IgM (polyclonal goat F(ab′)₂ fragments to human IgM, MP Biomedicals, Santa Ana, CA) for the indicated time periods.

Takahashi K., Sivina M., Hoellenriegel J., Oki Y., Hagemeister F.B., Fayad L., Romaguera J.E., Fowler N., Fanale M.A., Kwak L.W., Samaniego F., Neelapu S., Xiao L., Huang X., Kantarjian H., Keating M.J., Wierda W., Fu K., Chang W.C., Vose J.M., O’Brien S., Davis R.E, & Burger J.A. (2015). CCL3 and CCL4 are biomarkers for BCR pathway activation and prognostic serum markers in diffuse large B cell lymphoma (DLBCL). British journal of haematology, 171(5), 726-735.

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Characterization of AML Mononuclear Cells

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Frozen aliquots of Ficoll-isolated mononuclear cells from pretreatment (‘diagnostic') peripheral blood or bone marrow specimens from adult patients with AML were obtained from repositories at the Fred Hutchinson Cancer Research Center. We used the 2008 WHO criteria to define AML^{12 (link)} and the refined United Kingdom Medical Research Council criteria to assign cytogenetic risk.^{13 (link)} Patients provided written informed consent for the collection and use of their biospecimens for research purposes under protocols approved by the Fred Hutchinson Cancer Research Center Institutional Review Board. Clinical data were de-identified in compliance with Health Insurance Portability and Accountability Act regulations. Cells were cultured in Iscoves' Modified Dulbecco's medium (Life Technologies, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (HyClone, Thermo Scientific, Logan, UT, USA) and 10 ng/ml each of interleukin-3, stem cell factor, granulocyte-colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (all from Life Technologies).

Laszlo G.S., Gudgeon C.J., Harrington K.H, & Walter R.B. (2015). T-cell ligands modulate the cytolytic activity of the CD33/CD3 BiTE antibody construct, AMG 330. Blood Cancer Journal, 5(8), e340-.

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Cytokine Profiling of Cell Models

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Dulbecco's Modified Eagle Medium (DMEM); penicillin, streptomycin (Thermo Fisher Scientific); fetal bovine serum (Euroclone); Iscove's Modified Dulbecco's Medium (IMDM) (Life Technologies); TruSeq RNA Sample Prep Kit v2 (Illumina); NucleoSpin RNA II (Macherey-Nagel); MuLV Reverse Transcriptase (Applied Biosystems); Flowcytomix Multiple Analyte Detection Kit (eBioscience); Human Inflammatory Cytokines Multi-Analyte ELISArray™ Kit (Qiagen); Human IL6 DuoSet and the Human CXCL8/IL8 Quantikine ELISA Kits (R&D Systems Inc); MMP11 ELISA kit (Emelca Bioscience); ELISA-based TransAM NFkB Family kit (Active Motif); transwell for migration assay (Costar, Corning); EGF, bFGF, IL6 and IL8 (PeproTech); anti-human Ki67 monoclonal antibody (MAb), diaminobenzidine (DAB), (Dako); anti-human vimentin (clone V9, Santa Cruz); anti-human alpha smooth muscle actin, α-SMA (clone 1A4, R&D System), anti-human Ki67 (clone MIB-1, Dako); anti-human LAMP2 (#HPA029100) and anti-human NF-κB1 (#HPA027305, Sigma-Aldrich); anti-human NF-κB p65 RelA (#8242, Cell Signalling); Tocilizumab (Roche); Matrigel (BD Biosciences); all other reagents were obtained from Sigma-Aldrich.

Avnet S., Di Pompo G., Chano T., Errani C., Ibrahim-Hashim A., Gillies R.J., Donati D.M, & Baldini N. (2017). Cancer-associated mesenchymal stroma fosters the stemness of osteosarcoma cells in response to intratumoral acidosis via NF-κB acivation. International journal of cancer, 140(6), 1331-1345.

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Generating Liver Organoid from hiPSCs

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HLCs were differentiated from hiPSCs according to a published protocol [7 (link)], with minor modifications.
To generate hiPSC-LOs, hiPSC endoderm cells (250,000 cells), con-ECs (175,000 cells), and con-MCs (25,000 cells) or UC-derived ECs (UC-EC) (175,000 cells) and MCs (UC-MC) (25,000 cells) were cocultured in serum-free differentiation (SFD) medium containing epidermal growth factor (EGF, 10 ng/ml; Sigma-Aldrich), vascular endothelial growth factor (VEGF, 10 ng/ml; Life Technologies, Carlsbad, CA, USA), basic fibroblast growth factor (bFGF, 10 ng/ml; Wako Pure Chemical Industries), hepatocyte growth factor (HGF, 20 ng/ml; Sigma-Aldrich), and dexamethasone (100 nM; Sigma-Aldrich) in a three-dimensional (3D) microwell plate (Kuraray, Tokyo, Japan). The SFD medium contained 375 ml Iscove’s modified Dulbecco’s medium (Life Technologies), 125 ml Ham’s F-12 K medium (Life Technologies), 5 ml B27 supplement (Life Technologies), 2.5 ml N2 supplement (Life Technologies), 0.05% bovine serum albumin (Sigma-Aldrich), 2 mM l-glutamine (Life Technologies), 1% penicillin–streptomycin (Life Technologies), 0.45 mM monothioglycerol solution (Wako Pure Chemical Industries), and 0.5 mM l-ascorbic acid (Sigma-Aldrich). The hepatic lineage cells and LOs were differentiated and maintained at 37 °C in a humidified incubator with 5% CO₂.

Nie Y.Z., Zheng Y.W., Ogawa M., Miyagi E, & Taniguchi H. (2018). Human liver organoids generated with single donor-derived multiple cells rescue mice from acute liver failure. Stem Cell Research & Therapy, 9, 5.

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Generation of Human and Mouse Macrophages

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To generate HMDM, CD14⁺ monocytes were isolated from human buffy coats, obtained from the Australian Red Cross Blood Service. Peripheral blood mononuclear cells were isolated using Ficoll (GE Healthcare) density gradient separation followed by positive selection of monocytes (CD14⁺ magnetic beads), as previously described (17 (link)). Monocytes were then cultured in Iscove’s modified Dulbecco’s medium (Life Technologies) for 7 d to generate HMDM. HMDM media was supplemented with 10% heat-inactivated FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin (Life Technologies), as well as 1 × 10⁴ U/mL recombinant human colony stimulating factor-1 (a gift from Chiron). penicillin and streptomycin were excluded from cultures during bacterial infection assays. To generate mouse BMM, bone marrow cells from the femurs and tibias of C57BL/6 mice were cultured for 7 d in the presence of 1 × 10⁴ U/mL recombinant human colony stimulating factor-1, as previously described (69 (link)).

Stocks C.J., Phan M.D., Achard M.E., Nhu N.T., Condon N.D., Gawthorne J.A., Lo A.W., Peters K.M., McEwan A.G., Kapetanovic R., Schembri M.A, & Sweet M.J. (2019). Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immune-mediated zinc toxicity for dissemination. Proceedings of the National Academy of Sciences of the United States of America, 116(13), 6341-6350.

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Isolation and Maintenance of Hematopoietic Cell Lines

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EOL-1 cells carried the WT F/P fusion oncogene (Braunschweig, Germany) [49 (link)]. Ba/F3 cells expressing T674I F/P resistant to imatinib (IR) have been described previously [1 ]. After obtaining blood from the above-mentioned patients, polymorphonuclear leucocytes were separated by standard laboratory procedures [50 (link)]. Eosinophils were then separated by depletion of neutrophils with anti-CD16-coated magnetic microbeads using the magnetic cell separation system (MACS;Miltenyi Biotec GmbH, Bergisch- Gladbach, Germany). All these cell lines and primary cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA) at 37°C in a humidified atmosphere of 5% CO2. IL-3-dependent 32D cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum(FCS) and 10% WEHI-3 conditioned medium as a source of IL-3 [51 (link)]. MACS immunomagnetic cell separation (Miltenyi Biotech, Auburn, CA) using a hapten-conjugated antibody against CD34, which was coupled to beads, was used to isolate CD34+ cells. CD34+ cells were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Paisley, United Kingdom) supplemented with 8% FCS, 50 Amol/L of h-mercaptoethanol, 10 units/mL of penicillin, 10 Ag/mL of streptomycin, and 2 mmol/L of glutamine at a density of 0.3 × 10⁶ cells/mL.

Li B., Zhang G., Li C., Li R., Lu J., He Z., Wang Q., Peng Z., Wang J., Dong Y., Zhang C., Tan J.Q., Bahri N., Wang Y, & Duan C. (2016). Lyn mediates FIP1L1-PDGFRA signal pathway facilitating IL-5RA intracellular signal through FIP1L1-PDGFRA/JAK2/Lyn/Akt network complex in CEL. Oncotarget, 8(39), 64984-64998.

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Culturing Hematopoietic Stem Cells

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Kasumi-1, 293T and U937 cells were cultured as previously described.^{14 (link)} G-CSF (granulocyte colony-stimulating factor)-mobilized peripheral blood CD34+ cells were cultured in Iscove's modified Dulbecco's medium (Life Technologies, Karlsruhe, Germany) supplemented with 20% fetal calf serum, 20 ng/ml Flt-3 l, 20 ng/ml GM-CSF, 20 ng/ml stem cell factor (SCF), 20 ng/ml thrombopoietin, 20 ng/ml interleukin (IL)-6, 10 ng/ml IL-3 (all cytokines were obtained Peprotech, Hamburg, Germany), 100 U/ml penicillin/streptomycin and 2 mM L-glutamine. Retroviral transduction and long-term cultivation were performed as previously described.^{5 (link)} IMR-90 cells (ATCC-CCL-186) were cultured as suggested by the supplier.

Wichmann C., Quagliano-Lo Coco I., Yildiz Ö., Chen-Wichmann L., Weber H., Syzonenko T., Döring C., Brendel C., Ponnusamy K., Kinner A., Brandts C., Henschler R, & Grez M. (2014). Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors. Leukemia, 29(2), 279-289.

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