Lineage cell depletion kit

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The Lineage Cell Depletion Kit is a laboratory instrument used for the selective depletion of unwanted cell populations from heterogeneous samples. It facilitates the isolation and enrichment of target cell populations by removing specific lineage-committed cells.

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Lineage depletion kit (36 citations)

159 protocols using lineage cell depletion kit

Bone Marrow Transplantation and Flow Cytometry

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Bone marrow cells were harvested from BALB/c mice (The Jackson Lab, #000651), Flt3^ITD and Flt3^ITD;Tet2^+/− transgenic BALB/c mice^{33 (link)} (a kind gift from Evan Lind’s lab, Oregon Health & Science University, Portland, OR). All mouse work was performed with approval from the Oregon Health & Science University Institutional Animal Care and Use Committee. For the BM transduction experiment, BM lineage-negative cells were enriched using Lineage Cell Depletion Kit (#130–090–858, Miltenyi Biotec), cultured overnight in medium containing IL-3, IL-6, and stem cell factor, and infected with retrovirus expressing FLT3 or IDH1 WT and mutants. Cytokines are purchased from PepreTech. A total of 2000 lineage-negative cells per well were seeded into 6-well plate with 1.1 mL of Methocult M3534 methylcellulose medium (StemCell Technologies) for 10 days. Images were taken and colonies (>50 cells) were counted via STEMvision™ colony counting software (StemCell Technologies). Colony cells were harvested, stained with cell surface markers and analyzed by FACS.

Zhang H., Savage S., Schultz A.R., Bottomly D., White L., Segerdell E., Wilmot B., McWeeney S.K., Eide C.A., Nechiporuk T., Carlos A., Henson R., Lin C., Searles R., Ho H., Lam Y.L., Sweat R., Follit C., Jain V., Lind E., Borthakur G., Garcia-Manero G., Ravandi F., Kantarjian H.M., Cortes J., Collins R., Buelow D.R., Baker S.D., Druker B.J, & Tyner J.W. (2019). Clinical resistance to crenolanib in acute myeloid leukemia due to diverse molecular mechanisms. Nature Communications, 10, 244.

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In Vivo T-cell Reconstitution Assay

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The procedure for in vivo T-cell reconstitution was based on previous publications.⁴⁸ (link) Bone marrow lineage negative (Lin^-) cells from Dhx15^fl/fl or Dhx15 KO (Lck-cre Dhx15^fl/fl) mice were enriched using Lineage Cell Depletion Kit (Miltenyi Biotec), and pre-stimulated for 24 hr in DMEM (Gibco) containing 20% FBS (Gibco), 1% penicillin/streptomycin (Hyclone), 20 ng/mL mFLT3-L, 20 ng/mL mTPO and 100 ng/mL mSCF (PeproTech). Cells were then transduced with MigR1-Myc or MigR1 retroviruses (details in “Lentiviral and retroviral transduction”), and transplanted into irradiated (1.5 Gray) female NOD.Cg-Prkdc^scid Il2rg^tm1Vst/Vst (NPG) mice. T-cell lineage reconstitution was assessed 7 weeks after transplant in peripheral blood by flow cytometry.

Li Q., Guo H., Xu J., Li X., Wang D., Guo Y., Qing G., Van Vlierberghe P, & Liu H. (2023). A helicase-independent role of DHX15 promotes MYC stability and acute leukemia cell survival. iScience, 27(1), 108571.

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Generation of MLL-AF9 and MLL-ENL Mouse Models

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Flt3^ITD/+ mice26 (link) were kindly provided by Gary Gilliland and crossed with Rosa26^Cas9/+ mice6 (link). Freshly isolated bone marrow from 6- to 10-week-old female Rosa26^Cas9/+, Flt3^ITD/+; Rosa26^Cas9/+ or moribund Npm1^flox−cA/+; Flt3^ITD/+ mice was used. Bone marrow cells were exposed to erythrocyte lysis (BD PharmLyse, BD Bioscience), followed by magnetic bead selection of Lin^- cells using the Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Lin^- cells were cultured in X-VIVO 20 (Lonza) supplemented with 5% BIT serum substitute (Stem Cell Technologies), 10ng ml^-1 IL3 (Peprotech), 10ng ml^-1 IL6 (Peprotech) and 50ng ml^-1 of SCF (Peprotech). Retrovirus constructs pMSCV-MLL-AF9-IRES-YFP and pMSCV-MLL-ENL-IRES-Neo were used with package plasmid psi-Eco to produce retrovirus. 293T cells (Life Technologies) were cultured and prepared for transduction in 10cm plates as described above. For virus production, 5 μg of the above plasmids and 5 μg psi-Eco packaging vector were transfected drop wise into the 293T cells using 47.5 μl TransIT LT1 (Mirus) and 600 μl Opti-MEM (Invitrogen). Transduction of primary mouse cells was performed in 6-well plates as mentioned above. After transduction, cells were sorted for YFP (for MLL-AF9) or selected with neomycin (for MLL-ENL).

Barbieri I., Tzelepis K., Pandolfini L., Shi J., Millán-Zambrano G., Robson S.C., Aspris D., Migliori V., Bannister A.J., Han N., De Braekeleer E., Ponstingl H., Hendrick A., Vakoc C.R., Vassiliou G.S, & Kouzarides T. (2017). Promoter-bound METTL3 maintains myeloid leukaemia via m6A-dependent translation control. Nature, 552(7683), 126-131.

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Isolation and Purification of Hematopoietic Progenitors

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Tibias and femurs were crushed in PBS containing 3% of fetal calf serum (FCS). Red blood cells were lysed using ACK buffer (Gibco). Bone marrows were depleted in mature cells, expressing the lineage markers Cd5, B220, Cd11b, Gr-1, Ter-119 or 7-4, using the lineage cell depletion kit (Miltenyi Biotec). GMPs (Lineage⁻, Cd45⁺, C-Kit⁺, Sca-1⁻, Cd34⁺, FcγR⁺), LK (Lineage⁻, Cd45⁺, C-Kit⁺, Sca-1⁻), LSK (Lineage⁻, Cd45⁺, C-Kit⁺, Sca-1⁺) were purified using the FACS Aria III cell sorter (Beckman Dickinson). Antibodies used for GMP staining are described in supplemental materials.

Poplineau M., Vernerey J., Platet N., N’guyen L., Hérault L., Esposito M., Saurin A.J., Guilouf C., Iwama A, & Duprez E. (2019). PLZF limits enhancer activity during hematopoietic progenitor aging. Nucleic Acids Research, 47(9), 4509-4520.

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Lentiviral Knockdown of FoxO1 in Hematopoietic Stem Cells

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pGIPZ lentiviral mouse FoxO1 shRNAs were obtained from Open Biosystems, and the SF-LV-shRNA-EGFP vector was generously provided by Dr. Rudolph [25] (link). FoxO1 shRNA was cut from the pGIPZ vector by XhoI and MluI and then cloned into an SF-LV lentiviral vector. The plasmids SF-LV-shFoxO1-EGFP, pSPAX2 and pMD2.G were co-transfected into the packaging cell line 293T using Lipofectamine 2000 (Invitrogen) to generate the virus. Virus supernatants were harvested 48 and 72 h after transfection. Lin⁻c-Kit⁺ cells from the BM of Rictor-deleted mice were enriched using a Lineage Cell Depletion Kit and c-Kit (CD117) magnetic beads (MiltenyiBiotec) according to the manufacturer's protocol. The Lin⁻c-Kit⁺ cells were then transduced with viruses, and the transduction efficiency was measured using flow cytometry. BM mononucleated cells (BMMNCs) from 8-week-old B6.SJL mice (CD45.1⁺) with virus-transduced Lin⁻c-Kit⁺ cells were transplanted into lethally irradiated (9.6 Gy) B6.SJL mice.

Zhang Y., Hu T., Hua C., Gu J., Zhang L., Hao S., Liang H., Wang X., Wang W., Xu J., Liu H., Liu B., Cheng T, & Yuan W. (2014). Rictor Is Required for Early B Cell Development in Bone Marrow. PLoS ONE, 9(8), e103970.

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Isolation of Murine Hematopoietic Progenitors

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For in vitro experiments, total bone marrow was flushed out from femurs and tibia of 8-week-old wild-type C57BL/6 mice. The lineage-negative fraction was purified using the Lineage Cell Depletion Kit (Miltenyi Biotec, Calderara di Reno, Italy), according to manufacturer’s protocol. Lineage-depleted (Lin−) bone marrow progenitor cells were plated and cultured as previously described.^{16 (link)}

Dell'Aversana C., Giorgio C., D'Amato L., Lania G., Matarese F., Saeed S., Di Costanzo A., Belsito Petrizzi V., Ingenito C., Martens J.H., Pallavicini I., Minucci S., Carissimo A., Stunnenberg H.G, & Altucci L. (2017). miR-194-5p/BCLAF1 deregulation in AML tumorigenesis. Leukemia, 31(11), 2315-2325.

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Metabolic Profiling of Bone Marrow Cells

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Single cell suspensions were prepared from bone marrow. Cells were then fractionated into Lin^- and Lin⁺ cells using a Lineage Cell Depletion Kit (Miltenyi Biotec), according to the manufacturer’s protocols. For measurement of OXPHOS, 10⁶ Lin^- and Lin⁺ cells were resuspended in XF assay medium (pH 7.4) containing GlutaMax (2 mM), sodium pyruvate (1 mM) and glucose (25 mM), plated onto Seahorse Bioscience XF24 cell culture plates coated with Cell Tak (BD Bioscience), and incubated without CO₂ at 37°C. Oxygen consumption rate (OCR) was measured using the Seahorse XF24 Analyzer (Agilent Technologies, Santa Clara, CA) in the presence or absence of Oligomycin (0.6 μM), FCCP (1 μM), and Antimycin (1 μM) and Rotenone (1 μM). For measurement of glycolysis, 10⁶ Lin^- and Lin⁺ cells were resuspended in assay medium (pH 7.4) (Sigma-Aldrich) containing L-Glutamine (2 mM), plated onto XF24 cell culture plates, and incubated as for OCR. Extracellular acidification rate (ECAR) was measured using the Seahorse XF24 Analyzer in the presence or absence of Glucose (10 mM), Oligomycin and 2-DG [20 (link), 21 (link)].

Kalim K.W., Zhang S., Chen X., Li Y., Yang J.Q., Zheng Y, & Guo F. (2017). mTOR has a developmental stage-specific role in mitochondrial fitness independent of conventional mTORC1 and mTORC2 and the kinase activity. PLoS ONE, 12(8), e0183266.

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Generating NOTCH1-Induced T-ALL Tumors

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To generate NOTCH1-induced T-ALL tumors in mice, we performed retroviral transduction of bone marrow cells enriched in Lineage negative cells isolated from female mice using magnetic beads (Lineage Cell Depletion Kit, Miltenyi Biotec) with an activated form of the NOTCH1 oncogene (ΔE-NOTCH1)^{10 (link)} and transplanted them via intravenous injection into lethally irradiated female isogenic recipients as previously described^{55 (link)}.

Herranz D., Ambesi-Impiombato A., Palomero T., Schnell S.A., Belver L., Wendorff A.A., Xu L., Castillo-Martin M., Llobet-Navás D., Cardo C.C., Clappier E., Soulier J, & Ferrando A.A. (2014). N-Me, a long range oncogenic enhancer in T-cell acute lymphoblastic leukemia. Nature medicine, 20(10), 1130-1137.

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Generating Dendritic Cells from Bone Marrow

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Lin^neg BM cells isolated using Lineage cell depletion kit (Miltenyi, Auburn, CA) were cultured (5 × 10⁵ cells/well) for 5 d with GM-CSF/IL-4 (each 10 ng/ml) plus cytokines (10-20 ng/ml). Cultured cells were depleted of CD11c⁺/CD19⁺ cells (10-15% in total cells), and examined for % CGr1 cells and DC-HIL expression.

Chung J.S., Tamura K., Cruz PD J.r, & Ariizumi K. (2014). DC-HIL-expressing Myelomonocytic Cells Are Critical Promoters of Melanoma Growth. The Journal of investigative dermatology, 134(11), 2784-2794.

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Isolation and Analysis of Mouse Hematopoietic Stem Cells

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PB was collected from the tail vein and kept in Microvette tubes (Sarstedt) for further analysis. Blood parameters were analyzed on Sysmex XE-5000 (Sysmex Europe). For the donor contribution analysis, red blood cells were lysed with ammonium chloride (NH₄Cl; STEMCELL Technologies) and stained with antibodies. BM cells were isolated by crushing tibias, femurs, and iliac bones in a mortar with pestle. For LSK (Lineage⁻ SCA-1⁺ c-KIT⁺) sorting, BM cells from spine and sternum were also collected. Single-cell suspensions were prepared by filtering through a 40 μm nylon cell strainer (Fisher Scientific). Lin⁻ cells were obtained using a lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Miharada N., Rydström A., Rak J, & Larsson J. (2021). Uncoupling key determinants of hematopoietic stem cell engraftment through cell-specific and temporally controlled recipient conditioning. Stem Cell Reports, 16(7), 1705-1717.

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