Interleukin 3 (il 3)

Manufactured by STEMCELL

Sourced in Canada, United States, United Kingdom

IL-3, or Interleukin-3, is a cytokine that plays a key role in the growth and differentiation of various blood cell types, including hematopoietic stem cells, myeloid progenitor cells, and lymphoid progenitor cells. It is a critical regulator of cell proliferation and survival within the hematopoietic system.

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Lab products found in correlation

Stem cell factor (scf), by STEMCELL (36 mentions) Interleukin 6 (il 6), by STEMCELL (33 mentions) Gm csf, by STEMCELL (17 mentions) Flt3l, by STEMCELL (13 mentions) G csf, by STEMCELL (7 mentions) Stemspan, by STEMCELL (7 mentions) Stemspan sfem, by STEMCELL (6 mentions) Erythropoietin, by STEMCELL (6 mentions) Flt 3 ligand, by STEMCELL (5 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (4 mentions) Fetal bovine serum (fbs), by STEMCELL (4 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (4 mentions) Glutamax, by Thermo Fisher Scientific (4 mentions) Stem cell factor (scf), by R&D Systems (3 mentions) Methocult h4434, by STEMCELL (3 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Il 23, by R&D Systems (2 mentions) Interleukin 7 (il 7), by R&D Systems (2 mentions) Methocult 3234, by STEMCELL (2 mentions) Interleukin 7 (il 7), by STEMCELL (2 mentions)

69 protocols using interleukin 3 (il 3)

Expansion of Human Umbilical Cord Blood CD34+ Cells

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CD34⁺ cells were purified from human umbilical cord blood (UCB, provided from normal full-term deliveries after informed consent as approved by the Maternal and Child Health Hospital of Hunan Province, Hunan, China) by positive selection using the CD34⁺ magnetic selective beads system (Miltenyi Biotec, Germany) according to the manufacturer's instructions. The purification of CD34⁺ cells were checked by CD34 antibody (BD) staining and flow cytometry analysis. Cells were first cultured at 10⁵ cells/ml for 6 days in Serum Free Expansion Medium (SFEM, Stem Cell Technologies) supplemented with 10% fetal bovine serum (FBS, Stem Cell Technologies), 10 ng/ml stem cell factor (SCF), 1 ng/ml IL-3, and 1 IU/ml erythropoietin (Stem Cell Technologies) at 37°C in 5% CO₂, and then cultured for 8 more days in the above complete medium with the presence of 30% FBS and the absence of IL-3 and SCF.

Sun Z., Wang Y., Han X., Zhao X., Peng Y., Li Y., Peng M., Song J., Wu K., Sun S., Zhou W., Qi B., Zhou C., Chen H., An X, & Liu J. (2015). miR-150 inhibits terminal erythroid proliferation and differentiation. Oncotarget, 6(40), 43033-43047.

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Culturing Primary Hematopoietic Cells

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Primary Ph⁺ ALL bone marrow cells were kindly provided by Dr Michael Caligiuri (Ohio State University, Columbus, OH) and Dr Martin Carroll (University of Pennsylvania, Philadelphia, PA). Cells were seeded on a substrate of adherent, mitomycin C-treated OP9 stromal cells and maintained in SFEM (Stem Cell Technology, Vancouver, Canada) supplemented with IL-3 (10 ng/ml), IL-7 (10 ng/ml), FLT3L (20 ng/ml) and SCF (30 ng/ml) (ProSpec, Israel).
CD34⁺ CML cells were kindly provided by Dr Tessa Holyoake (University of Glasgow, United Kingdom) and cultured in SFEM supplemented with IL-3 (20 ng/ml), IL-6 (20 ng/ml), SCF, and thrombopoietin (10 ng/ml).
Commercially purchased (Stem Cell Technologies) cord blood CD34⁺ cells were cultured in SFEM (Stem Cell Technologies) enriched with the CC100 cytokine cocktail (SCF, 100 ng/ml; FLT3L, 100 ng/ml; IL-3, 20 ng/ml; IL-6, 20 ng/ml).
Ph⁺ and normal CD34⁺ primary cells were kept in culture at 37 °C, under a 5% CO₂ humidified atmosphere. Cell counts were performed using 0.4% Trypan Blue Solution and a Neubauer hemocytometer.

Riva B., Dominici M.D., Gnemmi I., Mariani S.A., Minassi A., Minieri V., Salomoni P., Canonico P.L., Genazzani A.A., Calabretta B, & Condorelli F. (2016). Celecoxib inhibits proliferation and survival of chronic myelogeous leukemia (CML) cells via AMPK-dependent regulation of β-catenin and mTORC1/2. Oncotarget, 7(49), 81555-81570.

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Establishing Fancd2 Mouse Cell Lines

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Non-adherent cells were harvested from Fancd2^+/+, Fancd2^+/− and Fancd2^−/− mouse LTBMC at week 4 and cultured in six-well tissue culture plates in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 20% fetal calf serum (FCS) and 1.0 ng/mL Interleukin-3 (IL-3) (Peprotech, Rocky Hill, NJ). The Fancd2^+/+, Fancd2^+/− and Fancd2^−/− cell lines were passaged weekly for 10 weeks to establish primary IL-3-dependent cell lines using published methods (14 (link), 15 (link)).
Clonal cell sublines were established from each of the Fancd2^+/+, Fancd2^+/− and Fancd2^−/− parent lines by expansion of single colonies. Cells from primary IL-3-dependent cell lines were plated in 0.8% methylcellulose supplemented with 10% IMDM, 30% fetal bovine serum (FBS), 1% bovine serum albumin, 2 ng/mL IL-3 (Stemcell Technologies, Vancouver, Canada) at variable cell densities. At day 14, individual colonies were harvested and each cultured in a well of a 96-well plate in 0.2 mL of IMDM supplemented with 30% FBS and 1 ng/mL of IL-3. Cells were then replated in methylcellulose, colonies selected at 14 days and cultured as above, to establish subcloned lines. Confirmation of genotype after repeated subcloning was carried out for each cell line.

Berhane H., Epperly M.W., Goff J., Kalash R., Cao S., Franicola D., Zhang X., Shields D., Houghton F., Wang H., Wipf P., Parmar K, & Greenberger J.S. (2014). Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2−/−) Mice. Radiation research, 181(1), 76-89.

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Establishing Fancd2 Mutant Cell Lines

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Nonadherent cells were harvested from (FVB/N) Fancd2^+/+, Fancd2^+/− and Fancd2^−/− mouse LTBMC at week 4 and cultured in six-well tissue culture plates in Iscove's modified Eagles medium (IMDM) supplemented with 20% fetal calf serum (FBS) and 1.0 ng/mL Interleukin 3 (IL-3) (Peprotech, Rocky Hill, NJ). The Fancd2^+/+, Fancd2^+/− and Fancd2^−/− cell lines were passaged weekly for 10 weeks to establish primary IL-3-dependent cell lines (16 (link)).
Clonal cell sublines were established from each of the (FVB/N) Fancd2^+/+, Fancd2^+/− and Fancd2^−/− parent lines by expansion of single colonies. Cells from primary IL-3-dependent cell lines were plated in 0.8% methylcellulose supplemented with 10% IMDM, 30% fetal bovine serum (FBS), 1% bovine serum albumin, 2 ng/mL IL-3 (Stemcell Technologies, Vancouver, Canada) at variable cell densities. At day 14, individual colonies were harvested and each cultured in a well of a 96-well plate in 0.2 mL of IMDM supplemented with 30% FBS and 1 ng/mL IL-3. Cells were then replated in methylcellulose-containing medium, colonies selected at day 14 and cultured as above to establish subcloned lines. Confirmation of genotype after repeated subcloning was performed for each cell line as published previously (16 (link)).

Berhane H., Shinde A., Kalash R., Xu K., Epperly M.W., Goff J., Franicola D., Zhang X., Dixon T., Shields D., Wang H., Wipf P., Li S., Gao X, & Greenberger J.S. (2014). Amelioration of Radiation-Induced Oral Cavity Mucositis and Distant Bone Marrow Suppression in Fanconi Anemia Fancd2−/− (FVB/N) Mice by Intraoral GS-Nitroxide JP4-039. Radiation research, 182(1), 35-49.

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Genome editing of CD34+ HSPCs

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CD34+ cells were thawed into H3000 (Stem Cell Technologies) supplemented with 1% CC100 containing human recombinant FLT3L, SCF, IL-3, and IL-6 (Stem Cell Technologies). 24 h later 150,000 CD34+ HSPC cells were electroporated with precomplexed 20 μM Cas9 (NEB Engen Cas9 NLS) and 50 μM sgRNAs. Genome editing confirmed 24 h later by PCR. 48 h after electroporation bulk cells were plated in MethoCult H4434 containing SCF, IL-3, EPO, and GM-CSF (Stem Cell Technologies). Either DMSO or the SETD8 inhibitor UNC0379 was added to the MethoCult, as indicated. Colonies were counted 14 days later.

Myers J.A., Couch T., Murphy Z., Malik J., Getman M, & Steiner L.A. (2020). The histone methyltransferase Setd8 alters the chromatin landscape and regulates the expression of key transcription factors during erythroid differentiation. Epigenetics & Chromatin, 13, 16.

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CD34+ Cell Expansion with ST7

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CD34 + cells were isolated from de-identified cord blood or adult peripheral blood and cultured in semisolid media with minimal growth factor supplements (IL-3, G-CSF, Stem Cell Technologies, Vancouver B.C.), with or without ST7 or its enantiomers. Cord blood was generously provided by Dr. Ludy Dobrila from the National Cord Blood Program (Howard P. Milstein Cord Blood Center, New York Blood Center, New York, NY). Briefly, CD34⁺ cells were enriched by negative selection of non-CD34+ cells using specific antibodies (RosetteSep reagent, Stem Cell Technologies, Vancouver, BC, Canada), using a Ficoll-paque density gradient, and further enriched by positive selection using EasySep (Stem Cell Technologies). The enriched cells were cultured in H4230 medium (Stem Cell Technologies) containing 1% Methylcellulose in Iscove’s MDM, 30% fetal bovine serum, 2 mM L-glutamine, 1% Bovine Serum Albumin and 10^–4 M β-mercaptoethanol, with GM-CSF (20 ng/ml) and IL-3 (20 ng/ml). Cells were cultured in a humidified atmosphere with 5% CO₂, at 37°C and CFU-GM colonies were enumerated on day 14; treated cultures were compared to untreated controls from the same subject. Statistical tests were performed using the GraphPad Prism program; a level of 0.05 was considered significant.

Faller D.V., Castaneda S.A., Zhou D., Vedamony M., Newburger P.E., White G.L., Kosanke S., Plett P.A., Orschell C.M., Boosalis M.S, & Perrine S.P. (2016). An oral Hemokine™, α-methylhydrocinnamate, enhances myeloid and neutrophil recovery following irradiation in vivo. Blood cells, molecules & diseases, 63, 1-8.

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Mononuclear Cell Purification and Erythropoiesis

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Mononuclear cells from MPN patients were obtained over a Ficoll density gradient and CD34+ cells were purified by a double-positive magnetic cell sorting system (AutoMACS, Miltenyi Biotec). CD34+ cells were cultured 2 days in a liquid culture system stimulating erythropoiesis, in the presence of 50 ng ml⁻¹ recombinant human stem cell factor (Amgen, Thousand Oaks, CA, USA), 50 U ml⁻¹ interleukin-3 (Novartis, Basel, Switzerland) and 3 U ml⁻¹ erythropoietin (EPO; Orthobiotech, Paris, France). Cells were then transduced with lentiviral vector encoding shRNA-HSP27 or control vector (CTL). The CD34+/GFP+ were sorted 24 h later and 750–1000 cells were plated in duplicate in semi-solid conditions (methylcellulose) (H4230, Stem Cell Technologies) supplemented with interleukin-3, stem cell factor and EPO. BFU-E colonies were counted 14 days later and were plucked from methylcellulose for genotyping. The JAK2V617F mutational status was analysed by quantitative real-time PCR (qRT-PCR), using fluorescent competitive probes.

Sevin M., Kubovcakova L., Pernet N., Causse S., Vitte F., Villeval J.L., Lacout C., Cordonnier M., Rodrigues-Lima F., Chanteloup G., Mosca M., Chrétien M.L., Bastie J.N., Audia S., Sagot P., Ramla S., Martin L., Gleave M., Mezger V., Skoda R., Plo I., Garrido C., Girodon F, & de Thonel A. (2018). HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis. Nature Communications, 9, 1431.

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Isolation of Leukemic CD34+ Progenitors

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Immunomagnetic separation of bone marrow leukemic CD34-positive progenitors expressing the e14a3 BCR-ABL1 fusion was performed as previously described (Massimino et al., 2014 (link)). CD34-positive cells derived from healthy donors were obtained from ALLCELLS. CD34-positive cells were maintained in Stem Span SFEM supplemented with cytokines at low concentrations (Flt-3 ligand: 5 ng/ml, stem cell factor: 5 ng/ml, interleukin 3: 1 ng/ml, interleukin 6: 1 ng/ml) (all from Stem Cell Technologies).

Massimino M., Tirrò E., Stella S., Manzella L., Pennisi M.S., Romano C., Vitale S.R., Puma A., Tomarchio C., Di Gregorio S., Antolino A., Di Raimondo F, & Vigneri P. (2021). Impact of the Breakpoint Region on the Leukemogenic Potential and the TKI Responsiveness of Atypical BCR-ABL1 Transcripts. Frontiers in Pharmacology, 12, 669469.

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Isolation and Culture of CD34+ Hematopoietic Progenitors

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CD34⁺ cells were isolated from mononuclear cells by magnetic cell sorting using the EasySep human CD34-positive selection kit II (Stem Cell Technologies, Vancouver, Canada) following the manufacturer’s recommendations. CD34⁺ cells were plated at low-density in 2 to 4 wells of a SmartDish (Stem Cell Technologies) in MethoCult H4034 Optimum Methylcellulose-based media containing Stem Cell Factor, GM-CSF, G-CSF, interleukin-3, and EPO (Stem Cell Technologies). Cultures were placed in an incubator set at 37°C, 5% CO_2, and 90% humidity for 14 days. At the end of the incubation, colonies were scored (CFU-E, BFU-E, CFU-GM) in a blinded manner by 2 independent investigators, and photographs were documented with the STEMvision automated colony counter (Stem Cell Technologies).

Pollard J.A., Furutani E., Liu S., Esrick E., Cohen L.E., Bledsoe J., Liu C.W., Lu K., de Haro M.J., Surrallés J., Malsch M., Kuniholm A., Galvin A., Armant M., Kim A.S., Ballotti K., Moreau L., Zhou Y., Babushok D., Boulad F., Carroll C., Hartung H., Hont A., Nakano T., Olson T., Sze S.G., Thompson A.A., Wlodarski M.W., Gu X., Libermann T.A., D’Andrea A., Grompe M., Weller E, & Shimamura A. (2022). Metformin for treatment of cytopenias in children and young adults with Fanconi anemia. Blood Advances, 6(12), 3803-3811.

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Reprogramming Human PBMCs to iPSCs

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PBMCs were cultured in StemPro-34 serum-free medium (Stem Cell Technologies) supplemented with 100 ng/mL SCF (Stem Cell Technologies), 100 ng/mL FLT-3 (Stem Cell Technologies), 20 ng/mL IL-3 (Stem Cell Technologies), 20 ng/mL IL-6 (Stem Cell Technologies) prior to reprogramming with Cytotune™-iPS 2.0 Sendai virus (ThermoFisher Scientific) expressing OCT4, SOX2, KLF4 and cMYC (Bhatt et al. [38 (link)] and Chitrangi and Bhatt et al., [39 –41 ]), in the presence of 4 µg/ml Polybrene (EMD Millipore). Transduced cells were plated on vitronectin (Stem Cell Technologies) coated plates. iPSC colonies were mechanically cut and further expanded in mTeSR™ (Stem Cell Technologies). Human iPSCs were characterized by expression of Oct4, Nanog, Sox2, SSEA4 using flowcytometry and immunocytochemistry. These human iPSCs are suitable for directed differentiation, disease modeling, CRISPR-Cas9 mediated gene editing, developmental biology, drug discovery applications, organoid development and precision drug screening etc.

Chitrangi S., Vaity P., Jamdar A, & Bhatt S. (2023). Patient-derived organoids for precision oncology: a platform to facilitate clinical decision making. BMC Cancer, 23, 689.

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