Murine il 7

Manufactured by Thermo Fisher Scientific

Sourced in France

Murine IL-7 is a recombinant protein that represents the mouse interleukin-7 molecule. Interleukin-7 is a cytokine that plays a crucial role in the development and maintenance of T cells and B cells.

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14 protocols using murine il 7

Establishment of Murine B-ALL Model

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For establishment of a murine B-ALL model by overexpressing the N-Myc oncogene in hematopoietic stem progenitor cells, an MSCV-N-Myc-IRES-GFP plasmid together with the pCL-ECO packaging plasmid were transfected into HEK293T cells, followed by collection of the supernatant containing retroviruses 48–72 h after transfection. Lin⁻ fetal liver cells were purified and spin infected with N-Myc retroviruses. The infected cells were then incubated in Stemspan medium (StemCell) in the presence of 10 ng/mL murine SCF (Peprotech) and 10 ng/mL murine IL-7 (Peprotech) for 2 days. A total of 1–3 × 10⁵ infected Lin- BM cells were transplanted into lethally irradiated (10 Gy) recipient mice by retro-orbital injection. GFP⁺ BM B-ALL cells collected from primary recipient mice were sorted and transplanted into lethally irradiated 6- to 8-week-old recipients. The frequency of GFP⁺ cells in the peripheral blood of recipient mice was analyzed by flow cytometric analysis at the indicated time points post-transplantation to evaluate B-ALL development. In another set of experiments, 5 × 10⁵ B-ALL cells were cultured in 12-well plates with 500 μL of StemSpan serum-free medium (STEMCELL Technologies) containing 10 ng/mL mouse SCF (Peprotech), 10 ng/mL murine IL-7 (Peprotech), and 30 μg of ANGPTL2-containing SEVs, ANGPTL2-mut#2-containing SEVs or control SEVs for 6 h before transplantation.

Huang D., Yuan Y., Cao L., Zhang D., Jiang Y., Zhang Y., Chen C., Yu Z., Xie L., Wei Y., Wan J, & Zheng J. (2023). Endothelial-derived small extracellular vesicles support B-cell acute lymphoblastic leukemia development. Cellular Oncology (Dordrecht), 47(1), 129-140.

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Mouse Bone Marrow Progenitor Assays

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Primary mouse clonogenic progenitor assays or colony forming units (CFU) assays were performed by plating 5×10⁵ unfractionated nucleated BM cells in methylcellulose M3434 (StemCell Technologies, Vancouver, Canada) in 1 ml duplicates. Colonies were counted and scored after 7 days of incubation using standard morphological criteria^{39 (link)}. To assay granulocyte/macrophage (GM-CFU), unfractionated nucleated BM cells (5×10⁵) were suspended in methylcellulose medium (M3231, StemCell Technologies) with 0.01 ng/ml murine GM-CSF (Peprotech, Rocky Hill, NJ, US) and plated in 1 ml duplicates. Colonies were counted after 7 days. To assay PreB-cell colonies (PreB-CFU), unfractionated nucleated BM cells (5×10⁵) were suspended in methylcellulose medium (M3231, StemCell Technologies) with 0.1, 1 and 10ng/ml murine IL-7 (Peprotech) and plated in 1-ml duplicates. Colonies were counted after 7 days. Representative images were taken with a Keyence BZ-X710 microscope (Keyence, Itasca, IL, US). Images were viewed with the Keyence BZ-X viewer and BZ-X analyzer. Image brightness and contrast were edited using Affinity Designer. To assess the effect of known chemical inhibitors on CFUs from either RoLoRiG^+/+Mx1CRE^- or RoLoRiG^+/+Mx1CRE⁺ mice, cells were plated in the absence or presence of U0126 MEK inhibitor (5μM), PI3K inhibitor GDC-0941 (0.1μM) or mTOR inhibitor rapamycin (0.1μM).

Karra L., Romero-Moya D., Ksionda O., Krush M., Gu Z., Mues M., Depeille P., Mullighan C, & Roose J.P. (2020). Increased baseline RASGRP1 signals Enhance Stem Cell Fitness during Native Hematopoiesis. Oncogene, 39(45), 6920-6934.

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Limiting Dilution Assay for Hematopoietic Progenitors

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Limiting dilution assays were adapted from^{55 (link)}. In brief, ST2 ^{6 (link)} or OP9 ^{56 (link)} stromal cells were plated at a concentration of 4000 cells per well in a 96-well flat-bottom plate one day prior to plating. One day later, the s.e.m.i-confluent stromal cells were γ-irradiated with 2000 rad using a Cobalt source (Gammacell 40, Atomic Energy of Canada, Ltd). Populations of interest were sorted and plated at different concentrations (3, 6, 12, 24, or 48 cells per well). Cultures were maintained in supplemented IMDM, for ST2 co-cultures, or Opti-MEM (Gibco) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 ng/mL murine IL-7 (PeproTech), 50 ng/mL human FLT3-ligand (produced in-house) and 25 ng/mL murine stem cell factor (produced in-house) for OP9 co-cultures, at 37°C in a humidified atmosphere containing 10% CO2 in the air. After 14 or 18 days in culture, for ST2 or OP9 co-cultures, respectively, wells were inspected under an inverted microscope, and wells containing colonies of more than 50 cells were scored as positive.

Klein F., Roux J., Cvijetic G., Rodrigues P.F., von Muenchow L., Lubin R., Pelczar P., Yona S., Tsapogas P, & Tussiwand R. (2022). Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors. Nature immunology, 23(4), 505-517.

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Limiting Dilution Assay for Hematopoietic Progenitors

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Murine CD19+ Cell Expansion Protocol

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CD19+ cells were isolated from murine total bone marrow using the EasySep positive-selection system (Stem Cell TechnologiesGrenoble, France). Cells were expanded in DMEM supplemented with 10% FBS (Hyclone, Illkirsh, France), 2 mM L-glutamine, 10 mM HEPES (pH 8), 1 mM sodium pyruvate, 55 µM ß-mercaptoethanol, 50 µg/mL gentamicin, and 1x Primocin (Invivogen, Toulouse, France) in the presence of 5 ng/mL murine IL-7 and 10 ng/mL murine SCF (PreproTech, Neuilly-Sur-Seine, France). Cells were replated every 2 days, maintaining a concentration of 2e6 cells/mL, and used for assays after 6 days of expansion.

Fruit C., Couly F., Bhansali R., Rammohan M., Lindberg M.F., Crispino J.D., Meijer L, & Besson T. (2019). Biological Characterization of 8-Cyclopropyl-2-(pyridin-3-yl)thiazolo[5,4-f]quinazolin-9(8H)-one, a Promising Inhibitor of DYRK1A. Pharmaceuticals, 12(4), 185.

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In Vitro Thymocyte Differentiation

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Thymocyte subpopulations of interest were purified by MACS depletion followed by cell sorting and then plated onto OP9-DL1 monolayers (30 (link)). The OP9-DL1 cells were inactivated with Mitomycin C (Stem Cell) immediately prior to use. 10³ sorted thymocytes were seeded per well in 96-well plates in αMEM (Thermo Fisher) supplemented with 20% FCS (Bovogen Biologicals), penicillin/streptomycin/gentamycin (Sigma), 2 ng/mL murine IL-7 (Peprotech), and 5 ng/mL human FLT3L (Peprotech). The media were refreshed every 2 days and freshly inactivated OP9-DL1 cells were added every 4 days.

Oh S., Liu X., Tomei S., Luo M., Skinner J.P., Berzins S.P., Naik S.H., Gray D.H, & Chong M.M. (2023). Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential. Frontiers in Immunology, 14, 1106652.

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In vitro T cell differentiation from fetal liver progenitors

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In vitro cultures of lineage marker-negative c-kit-positive fetal liver (FL) cells with stromal cells were described previously (Hozumi et al., 2003 (link); Abe et al., 2010 (link)). Briefly, lineage markers- (Lin; TER119, CD19, Mac-1, Gr-1) negative, c-kit-positive cells were isolated from E15.5 embryos and plated at 1 × 10⁴ cells on a monolayer of OP9 for T cell induction into CD25⁺ DN3 stage with serial expression of Dll for 7 days in the presence of 5 ng/ml murine IL7 (Peprotech, London, UK) and 5 ng/ml human Flt3L (Peprotech) in 24-well culture dishes or 5 × 10² cells on OP9 expressing Dll or its swapping mutants for T cell induction into DP stage for 13 days in the presence of 1 ng/ml IL7 and 5 ng/ml Flt3L. After cultures, growing cells were collected and analyzed for surface markers for 7 days (Lineage markers: Gr1, CD11b, CD11c, DX5 and ST2; CD45, Thy1.2, CD19, CD44 and CD25) or 13 days (CD45, CD19, Thy1.2, CD4 and CD8) cultures by flow cytometry. OP9 cell line was obtained from Dr. Yokoyama who published the report using this cell line (Yokoyama et al., 2013 (link)). We have confirmed the micoplasma-free status before the experiments.

Hirano K.I., Suganami A., Tamura Y., Yagita H., Habu S., Kitagawa M., Sato T, & Hozumi K. (2020). Delta-like 1 and Delta-like 4 differently require their extracellular domains for triggering Notch signaling in mice. eLife, 9, e50979.

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Culturing T-ALL and Leukemia Cells

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We performed cell culture of cell lines in standard conditions in a humidified atmosphere at 37°C under 5% CO₂. We obtained ALL-SIL (RRID: CVCL_1805), HPB-ALL (RRID: CVCL_1820) and JURKAT (RRID: CVCL_0065) T-ALL cell lines from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) cell line repository. We purchased HEK293T (RRID: CVCL_006) cells from American Type Culture Collection (ATCC).
We cultured primary mouse lineage negative bone marrow cells in Opti-MEM media (Life Technologies 51985091) supplemented with 10% FBS, 100 U mL⁻¹ penicillin G, 100 μg mL⁻¹ streptomycin, 55 μM β-mercaptoethanol, 10 ng mL⁻¹ IL3 (PeproTech 213-13), 10 ng mL⁻¹ IL6 (PeproTech 216-16), 25 ng mL⁻¹ IL7 (PeproTech 217-17), 50 ng mL⁻¹ SCF (PeproTech 250-03), and 50 ng mL⁻¹ Flt3L (PeproTech 250-31L). Primary mouse leukemia lymphoblasts in Opti-MEM media supplemented with 10% FBS, 100 U mL⁻¹ penicillin G, 100 μg mL⁻¹ streptomycin, 55 μM β-mercaptoethanol and 10 ng mL⁻¹ murine IL7 (Peprotech 217-17).

Belver L., Yang A.Y., Albero R., Herranz D., Brundu F.G., Quinn S.A., Pérez-Durán P., Álvarez S., Gianni F., Rashkovan M., Gurung D., Rocha P.P., Raviram R., Reglero C., Cortés J.R., Cooke A.J., Wendorff A.A., Cordó V., Meijerink J.P., Rabadan R, & Ferrando A.A. (2019). Gata3-controlled nucleosome eviction drives Myc enhancer activity in T-cell development and leukemia. Cancer discovery, 9(12), 1774-1791.

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Differentiation of B Cells from OP9-GFP Precursors

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OP9-GFP cells (12 (link)) were maintained
in MEMα (ThermoFischer) with 20% HI-FBS (Hyclone), glutamine,
β-mercaptoethanol, and penicillin/streptomycin. 1E4 sorted preproB cells
were added to 80–90% confluent OP9-GFP cells in 35 mm dishes in IMDM with
10% HI-FBS, 5 ng/ml murine FL, 1 ng/ml murine IL-7 (Peprotech), and
penicillin/streptomycin. Formation of mature myeloid and B lymphoid cells was
assessed using anti-CD11b-PE and anti-CD19-PerCP-Cy5.5 (1D3), and B cell
precursors were evaluated as done for marrow cells, in both cases gating on the
GFP^- population. Splenocytes were subjected to red blood cell
lysis and CD43-negative selection (Miltenyi). CD43^- cells were
cultured in RPMI with 15% HI-FBS, β-mercaptoethanol,
penicillin/streptomycin, and 20 ng/ml murine IL-4 (Peprotech) with either 10
μg/ml anti-murine CD40 (1C10, Biolegend) antibody or 25 μg/ml
E. coli O55:B5 LPS (Sigma), followed by analysis on day 4
using anti-B220-APC with anti-IgE-PE (RME-1, Biolegend) and anti-IgG1-BV421
(RMG1–1, Biolegend) or anti-IgG2b-PE (RMG2b-1, Biolegend). Serum obtained
by lancing the facial vein was analyzed for IgM, IgG, and IgA after 1:10,000
dilution using ELISA kits per the manufacturer’s instructions
(Invitrogen).

Guo H., Barberi T., Suresh R, & Friedman A.D. (2018). Progression from the Common Lymphoid Progenitor to B/Myeloid preproB and proB Precursors During B Lymphopoiesis Requires C/EBPα. Journal of immunology (Baltimore, Md. : 1950), 201(6), 1692-1704.

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Assessing T-cell Activation via STAT5 Signaling

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Cells of spleens, DLNs, and grafts from CTLA4-Fc/MR1–treated DEREG recipients day 100 after transplantation and naive mice were stimulated with murine IL-7 (PeproTech) or recombinant human IL-2 (Novartis) at a final concentration of 5 ng/mL or 320 ng/mL, respectively, for 5 minutes at 37°C in a water bath, then incubated for 25 minutes at 37°C with 5% CO₂. PBS was used as control on spleen and DLN samples. After stimulation, cells were fixed with CytoFix (BD) and permeabilized with Phosflow Perm Buffer III (BD) before staining with CD4, CD127, and STAT5 antibodies. Flow cytometry analysis was performed on BD FACSymphony.

Zhao Y., Nicholson L., Wang H., Qian Y.W., Hawthorne W.J., Jimenez-Vera E., Gloss B.S., Lai J., Thomas A., Chew Y.V., Burns H., Zhang G.Y., Wang Y.M., Rogers N.M., Zheng G., Yi S., Alexander S.I., O’Connell P.J, & Hu M. (2024). Intragraft memory-like CD127hiCD4+Foxp3+ Tregs maintain transplant tolerance. JCI Insight, 9(6), e169119.

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