Hsc003

Manufactured by R&D Systems

Sourced in Sweden

HSC003 is a laboratory equipment product manufactured by R&D Systems. It is designed for general laboratory use, but a detailed description of its core function is not available at this time.

Automatically generated - may contain errors

Lab products found in correlation

Hsc007, by R&D Systems (2 mentions) Retronectin coated plate, by Takara Bio (2 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions) Um171, by STEMCELL (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Polybrene, by Merck Group (2 mentions) Stemspan sfem, by STEMCELL (2 mentions) Gelcount system, by Oxford Optronix (1 mentions) Ic002p, by R&D Systems (1 mentions) L7914, by Merck Group (1 mentions) Psih1 h1 copgfp shrna vector, by System Biosciences (1 mentions) Cell resuspension solution, by R&D Systems (1 mentions) Cd90 bv421, by BD (1 mentions) Cd45ra fitc, by BD (1 mentions) Cd38 apc, by BD (1 mentions) Fcr blocking reagent, by Miltenyi Biotec (1 mentions) Myelocult m5300, by STEMCELL (1 mentions) Myelocult h5100, by STEMCELL (1 mentions) Axiocam 702, by Zeiss (1 mentions)

Close spelling variants of this product

HSC003 methylcellulose medium

14 protocols using hsc003

Hematopoietic Stem Cell Colony Assay

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Cells were treated for 18 hours in liquid culture and counted. Subsequently 2 × 10⁴ cells per mL were thoroughly mixed with pre-supplemented Methylcellulose media, without drug (HSC003, R & D Systems) and seeded into a 12-well plate at 250 μL per well. After 9 days colonies were stained with 25 μL of 8 mg/mL Iodonitrotetrazolium (Sigma-Aldrich) solution in pure ethanol. After overnight staining, colonies were imaged and counted using the GelCount system (Oxford Optronix, Abingdon, UK) using consistent settings for all plates.

Liberante F.G., Pouryahya T., McMullin M.F., Zhang S.D, & Mills K.I. (2015). Identification and validation of the dopamine agonist bromocriptine as a novel therapy for high-risk myelodysplastic syndromes and secondary acute myeloid leukemia. Oncotarget, 7(6), 6609-6619.

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Primary AML Colony Formation Assay

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Primary AML samples or normal mobilized peripheral blood samples were cultured with indicated drugs for 24 hours before being plated in human methylcellulose (R&D systems HSC003). Colonies were counts 10–17 days after the initial plating.

Jones C.L., Stevens B.M., Pollyea D.A., Culp-Hill R., Reisz J.A., Nemkov T., Gehrke S., Gamboni F., Krug A., Winters A., Pei S., Gustafson A., Ye H., Inguva A., Amaya M., Minhajuddin M., Abbott D., Becker M.W., DeGregori J., Smith C., D'Alessandro A, & Jordan C.T. (2020). Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells. Cell stem cell, 27(5), 748-764.e4.

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Hematopoietic Colony Assay for AML

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Primary AML samples or normal mobilized peripheral blood samples
were cultured without indicated metabolites or with indicated drugs for 24
hr before being plated in human methylcellulose (R&D systems HSC003).
Colonies were counts 10-17 days after the initial plating.

Jones C.L., Stevens B.M., D'Alessandro A., Reisz J.A., Culp-Hill R., Nemkov T., Pei S., Khan N., Adane B., Ye H., Krug A., Reinhold D., Smith C., DeGregori J., Pollyea D.A, & Jordan C.T. (2018). Inhibition of Amino Acid Metabolism Selectively Targets Human Leukemia Stem Cells. Cancer cell, 34(5), 724-740.e4.

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Lentiviral Knockdown of IL1RAP in Leukemia Cells

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For knockdown of IL1RAP, we cloned shRNA template oligonucleotides into the pSIH1-H1-copGFP shRNA vector (System Biosciences) and produced lentiviral particles as previously described (Barreyro et al., 2012 (link)). THP-1, HEL, or TF-1 cells were transduced with a nonsilencing control (luciferase) shRNA- or IL1RAP-specific shRNAs (MOI = 1), and transduced (GFP⁺) cells were FACS sorted before transplantation. For all shRNA transduction experiments, 8 µg/ml polybrene was added to cells before incubation with virus, and spin infection was performed after addition of virus (1,000 rcf for 1 h at 32°C) to facilitate transduction. Knockdown efficiency was measured by flow cytometry using anti–human IL-1RAcP/IL-1R3 APC or PE (FAB676A and FAB676P; R&D) and mouse IgG1 APC (17-4714; eBioscience) or PE (IC002P; R&D) as controls.
Primary human MDS and AML MNCs were transduced with IL1RAP shRNA lentiviruses (MOI = 1). 24 h after shRNA infection, GFP⁺ cells were FACS sorted and plated between 1.5 × 10⁵ and 2.5 × 10⁵ cells/ml in human complete methylcellulose medium (HSC003; R&D) supplemented with 40 µg/ml low-density lipoproteins (L7914; Sigma-Aldrich). Colonies were scored after 10–14 d.

Mitchell K., Barreyro L., Todorova T.I., Taylor S.J., Antony-Debré I., Narayanagari S.R., Carvajal L.A., Leite J., Piperdi Z., Pendurti G., Mantzaris I., Paietta E., Verma A., Gritsman K, & Steidl U. (2018). IL1RAP potentiates multiple oncogenic signaling pathways in AML. The Journal of Experimental Medicine, 215(6), 1709-1727.

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Clonogenic Assay for Hematopoietic Cells

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Bulk BM cells or sorted HSPCs/HSCs/pre-LSCs were spread in 1mL cytokine-containing methylcellulose media (HSC007 for mouse, HSC003 for human, R&D systems), and plated in 35mm culture dish. For murine cells, colonies were counted and replated after 7 days.

Ueda K., Kumari R., Schwenger E., Wheat J.C., Bohorquez O., Narayanagari S.R., Taylor S.J., Carvajal L.A., Pradhan K., Bartholdy B., Todorova T.I., Goto H., Sun D., Chen J., Shan J., Song Y., Montagna C., Xiong S., Lozano G., Pellagatti A., Boultwood J., Verma A, & Steidl U. (2021). MDMX acts as a pervasive preleukemic-to-acute myeloid leukemia transition mechanism. Cancer cell, 39(4), 529-547.e7.

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Colony-Forming Unit Assay Protocol

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The CFU-C assays were performed by plating 500 or 3000 CD34⁺ cells per plate in CFU-C media (R&D Systems, HSC003), according to the manufacturer’s instructions. Colonies consisting of at least 40 cells were counted after 15 days at 37 °C and 5% CO₂. CFU-C colonies were counted blindly regarding control and experimental samples.

Piragyte I., Clapes T., Polyzou A., Klein Geltink R.I., Lefkopoulos S., Yin N., Cauchy P., Curtis J.D., Klaeylé L., Langa X., Beckmann C.C., Wlodarski M.W., Müller P., Van Essen D., Rambold A., Kapp F.G., Mione M., Buescher J.M., Pearce E.L., Polyzos A, & Trompouki E. (2018). A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways. Nature Communications, 9, 3090.

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Hematopoietic Stem Cell Isolation and Culture

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Frozen CD34⁺ enriched cells were thawed and stored with FcR Blocking Reagent (Miltenyi Biotec) at 4 °C for 30 min. The cells were then stained as described above, with the following antibodies: CD34-phycoerythrin/Cy7, CD38-APC, CD45RA-FITC, and CD90-BV421 (1:25, BD Biosciences, San Jose, CA, US). After performing spectral compensation and setting the gates as mentioned earlier, the UCB HSC population was analyzed by flow cytometry (Table 1) and a total of 15000 viable CD34⁺ cells were collected from each sample using fluorescent activated cell sorting (FACS). The cells were mixed with Cell Resuspension Solution (R&D Systems, Oxon, Sweden) and Human Methylcellulose Complete Media containing EPO, Granulocyte macrophage colony-stimulating factor (GM-CSF), Interlukin-3 (IL-3), and Stem Cell Factor (SCF) (HSC003, R&D Systems). 500 cells/well were plated in triplicate in 6-well plates and placed in a humid chamber incubated at 37 °C with 5% CO2. Burst forming units-erythroid (BFU-Es) were counted in each well after 14 days of culture as indicated by R&D Systems (https://www.rndsystems.com/resources/protocols/human-colony-forming-cell-cfc-assay-using-methylcellulose-based-media).

Masoumi Z., Maes G.E., Herten K., Cortés-Calabuig Á., Alattar A.G., Hanson E., Erlandsson L., Mezey E., Magnusson M., Vermeesch J.R., Familari M, & Hansson S.R. (2019). Preeclampsia is Associated with Sex-Specific Transcriptional and Proteomic Changes in Fetal Erythroid Cells. International Journal of Molecular Sciences, 20(8), 2038.

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Evaluating Colony Formation in AML Cells

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Human methylcellulose complete media (HSC003, R&D systems) was selected to determine the function of FB23-2 on colony forming capacity in AML patient cells according to the manufacturer’s instruction. Briefly, 20,000 primary cells, with or without FB23-2 treatment, were suspended in 200 μl suspension buffer and seeded in 35 mm culture dishes with 1.5 ml methylceulose media. The cells were cultured under humid atmosphere in incubator for 12 days and the colony numbers were determined.

Huang Y., Su R., Sheng Y., Dong L., Dong Z., Xu H., Ni T., Zhang Z.S., Zhang T., Li C., Han L., Zhu Z., Lian F., Wei J., Deng Q., Wang Y., Wunderlich M., Gao Z., Pan G., Zhong D., Zhou H., Zhang N., Gan J., Jiang H., Mulloy J.C., Qian Z., Chen J, & Yang C.G. (2019). Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia. Cancer cell, 35(4), 677-691.e10.

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Quantifying LTC-IC Frequency in Mouse and Human

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To assess long-term culture-initiating cell (LTC-IC) frequency in mouse, serial dilutions of FACS-sorted LSK cells were plated in Myelocult M5300 (Stem Cell Technologies) mixed at 50:50 ratio with primary mouse stroma cell-conditioned medium as described above. To assess LTC-IC frequency in human, serial dilutions of mononuclear cells or immunomagnetically enriched CD34⁺ cells were plated in Myelocult H5100 (Stem Cell Technologies) mixed at 50:50 ratio with HS5-conditioned medium as described above. In each experiment, 10 technical replicates of each cell dose (indicated in the main text) were performed. After 4 (mouse) or 5 (human) weeks of culture, each well was assayed in HSC007 or HSC003 (R&D Systems), respectively in mouse and human, for the presence of CFU-C as described before (79 ). HSC frequency within the tested cell population was estimated using extreme limiting dilution analysis (ELDA) software (http://bioinf.wehi.edu.au/software/elda/) (80 (link)).

Kao Y.R., Chen J., Narayanagari S.R., Todorova T.I., Aivalioti M.M., Ferreira M., Ramos-Marques P., Pallaud C., Mantzaris I., Shastri A., Bussel J.B., Verma A., Steidl U, & Will B. (2018). Thrombopoietin receptor-independent stimulation of hematopoietic stem cells by eltrombopag. Science translational medicine, 10(458), eaas9563.

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Assessing Human LSCs and ILC1 Interactions

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Human LSCs were isolated from the blood of patients with AML and were cocultured with or without ILC1s isolated from the blood of healthy donors for 3 days. Cells were then plated into human methylcellulose complete media (R&D, HSC003) supplied with recombinant human SCF (50 ng/ml), human recombinant IL-3 (10 ng/ml), IL-6 (10 ng/ml), recombinant human GM-CSF (10 ng/ml), and recombinant human EPO (3 IU/ml). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 for 10–14 days. Colony numbers were counted using a microscope (Zeiss AxioCam 702).

Caligiuri M., Li Z., Ma R., Tang H., Zhang J., Marcucci G, & Yu J. (2023). Human ILC1s target leukemia stem cells and control development of AML. Research Square.

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