Macs lineage depletion kit, by Miltenyi Biotec - Product details

1

Generation of Bone-Marrow Chimeras

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For the generation of bone-marrow chimeras, irradiated NOD-LIRKO mice
(900 rad) were injected intravenously with lineage-depleted bone marrow cells
(5×10⁵ / mouse) isolated from NOD-Lox or NOD.BDC2.5 mice.
Lineage depletion was performed using magnetic separation with MACS
lineage-depletion kit (Miltenyi Biotech) according to the manufacturer’s
instructions. Lineage depletion efficiency was measured by Flow Cytometry.

Dirice E., Kahraman S., De Jesus D.F., El Ouaamari A., Basile G., Baker R.L., Yigit B., Piehowski P.D., Kim M.J., Dwyer A.J., Ng R.W., Schuster C., Vethe H., Martinov T., Ishikawa Y., Teo A.K., Smith R.D., Hu J., Haskins K., Serwold T., Qian W.J., Fife B.T., Kissler S, & Kulkarni R.N. (2019). Increased β-cell proliferation before immune cell invasion prevents progression of type 1 diabetes. Nature metabolism, 1(5), 509-518.

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2

Generation of Bone-Marrow Chimeras

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For the generation of bone-marrow chimeras, irradiated NOD-LIRKO mice
(900 rad) were injected intravenously with lineage-depleted bone marrow cells
(5×10⁵ / mouse) isolated from NOD-Lox or NOD.BDC2.5 mice.
Lineage depletion was performed using magnetic separation with MACS
lineage-depletion kit (Miltenyi Biotech) according to the manufacturer’s
instructions. Lineage depletion efficiency was measured by Flow Cytometry.

Dirice E., Kahraman S., De Jesus D.F., El Ouaamari A., Basile G., Baker R.L., Yigit B., Piehowski P.D., Kim M.J., Dwyer A.J., Ng R.W., Schuster C., Vethe H., Martinov T., Ishikawa Y., Teo A.K., Smith R.D., Hu J., Haskins K., Serwold T., Qian W.J., Fife B.T., Kissler S, & Kulkarni R.N. (2019). Increased β-cell proliferation before immune cell invasion prevents progression of type 1 diabetes. Nature metabolism, 1(5), 509-518.

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3

Hematopoietic Stem Cell-Derived Dendritic Cells

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To generate hematopoietic stem cell-DCs (Stem-DCs), lineage-negative cells were selected from BM-MNCs by magnetic beads (MACS lineage depletion kit, Miltenyi Biotech, Germany) as HSCs. HSCs were seeded at a concentration of 1 × 10⁶ cells per well in 24-well plates and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, granulocyte–macrophage colony-stimulating factor (GM-CSF, 100 ng/mL), stem cell factor (SCF, 50 ng/mL), and FMS-like tyrosine kinase 3 ligand (Flt3L, 50 ng/mL) (Peprotech, Rocky Hill, NJ, USA) for 7–10 days to promote their proliferation and differentiation into the monocyte lineage. Then, the cells were placed in culture media containing GM-CSF (1000 U/mL) and interleukin (IL)-4 (1000 U/mL) (Peprotech, USA) for 4–5 days to induce their differentiation into DCs.

Lee S.W., Lee H., Lee K.W., Kim M.J., Kang S.W., Lee Y.J., Kim H, & Kim Y.M. (2023). CD8α+ dendritic cells potentiate antitumor and immune activities against murine ovarian cancers. Scientific Reports, 13, 98.

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4

Calcium Flux Measurement in Lineage-Depleted Bone Marrow

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BM was freshly isolated and lineage depleted with the MACS Lineage Depletion Kit (Miltenyi Biotech, San Diego, CA). Cells were cultured for 30min in complete medium supplemented with 1µM Indo-1 prepared as stock supplemented with Pluronic-F127 and incubated at 37 °C for 30min. Cells were washed and stained for surface markers for 15min, washed and allowed to rest in for 15min PBS in PBS with Ca2⁺. FACS tubes were run at 37 °C in the sample port of the LSRII flow cytometer equipped with a 355nM excitation laser. Events were collected for 40 seconds prior to incubation with 25uM ATP or 1uM SDF1 to induce calcium transients. The average ratio, R, of bound/free Indo-1 (405nm/485nm emission) before simulation was used to determine baseline values. Identical samples were equilibrated in 10mM EDTA PBS w/o Ca2⁺ to determine R_min or stimulated with 1uM ionomycin to determine R_max. The Indo-1 dissociation constant (K_d) was assumed to be 237nM at 37 °C based on previous studies.⁴⁰ The following equation was then used to relate Indo-1 intensity ratios to [Ca_i²⁺] levels;

[{Ca}^{2 +}] = K_{d} \cdot \frac{(R - R_{min})}{(R_{max} - R)}

Luchsinger L.L., de Almeida M.J., Corrigan D.J., Mumau M, & Snoeck H.W. (2016). Mitofusin 2 maintains hematopoietic stem cells with extensive lymphoid potential. Nature, 529(7587), 528-531.

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5

Gamma Radiation Effects on Bone Marrow Transplants

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All mouse strains were bred from C57/BL6 mice to ensure consistency. Bone marrow transplants were performed on 5 animals of equal age, weight and sex ratios (1:1). National Institutes of Health guidelines for the care and use of laboratory animals and the recommendations of the ** laboratory animal ethics committee were followed throughout all animal research. The MACS lineage depletion kit (Miltenyi Biotec GmbH, Germany) was used to separate bone marrow cells from the femur and tibia bone marrow of an 8-week-old C57/BL6 mice. The procedure was carried out in accordance with the guidelines provided by the manufacturer. Using a Lenti-XTM concentrator (Clontech, Canada), we infected the recovered stem cells twice with either a highly purified form of the mice MALAT1 shRNA lentivirus or a scramble lentivirus.25 (link) To test the effects of a high dosage of gamma radiation, 9.6 Gy was administered to C57/BL6 mice that were around 8 weeks old. One million infected cells were injected retro-orbitally into the irradiated mice. Platelets were extracted from recipient mice 4–6 weeks after transplantation.

Sun Y., Wang T., Lv Y., Li J., Jiang X., Jiang J., Zhang D., Bian W, & Zhang C. (2022). MALAT1 promotes platelet activity and thrombus formation through PI3k/Akt/GSK-3β signalling pathway. Stroke and Vascular Neurology, 8(3), 181-192.

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6

Generating Bone-Marrow Chimeras in NOD Mice

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For the generation of bone-marrow chimeras, NOD Rag KO mice were irradiated (800 rad) and injected intravenously with lineage-depleted bone-marrow cells (5x10^5/mouse) from CD5 KD NOD mice. Alternatively, a mixture of lineage-depleted bone-marrow cells from NOD TCR KO and IgM KO mice (each either WT or CD5 KD) was injected into irradiated NOD Rag KO. All recipients were dox-treated. Lineage depletion was performed using magnetic separation with a MACS lineage-depletion kit (Miltenyi Biotech) according to the manufacturer’s instructions.

Schuster C., Kiaf B., Hatzihristidis T., Ruckdeschel A., Nieves-Bonilla J., Ishikawa Y., Zhao B., Zheng P., Love P.E, & Kissler S. (2022). CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells. Frontiers in Immunology, 13, 906499.

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7

Generating Myeloid Progenitor Cells from Bone Marrow

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BM-derived myeloid progenitor cells were generated from hematopoietic progenitor cells isolated from BM using a Miltenyi MACS lineage depletion kit. Cells were stimulated for 3 d in IL-3 (25 ng/ml), IL-6 (25 ng/ml), and SCF (50 ng/ml), washed, and stimulated for indicated times with rIFN-α4 (2,000 U/ml, PBL Assay Science). BMDMs were differentiated by incubating total BM cells in 15-cm tissue culture plates with recombinant M-CSF (100 ng/ml; Genentech) in DMEM for 7-10 d. M-CSF (100 ng/ml) was replenished every 3 d during culturing. BMDMs were plated overnight in M-CSF, rested for 4 h and stimulated for the indicated times with rIFN-α4 (2,000 U/ml; PBL Assay Science). For cell transfection experiments, 3T3 cells were transfected using Lipofectamine LTX with plus reagent (Life Technologies) according to manufacturer’s instruction.

Holmes D.A., Suto E., Lee W.P., Ou Q., Gong Q., Smith H.R., Caplazi P, & Chan A.C. (2015). Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling. The Journal of Experimental Medicine, 212(7), 1081-1093.

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8

Isolation and Characterization of Murine Hematopoietic Stem Cells

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Cells were labeled with the following of monoclonal antibodies purchased from eBioscience or Biolegend at the listed concentrations for 20–30 minutes, unless otherwise noted. CMP were isolated as described (4 (link)). Briefly, whole bone marrow was isolated and depleted of lineage positive cells by MACS lineage depletion kit (Miltenyi). Lineage negative cells were blocked with fluorescently-labeled anti-CD16/32 (93; 1:100), then incubated with biotinylated mAbs to CD45 (30-F11; 1:100), CD3 (17A2; 1:100), CD11b (M1/70; 1:600), CD11c (N418; 1:100), NK1.1 (PK136; 1:100), F4/80 (BM8; 1:100), B220 (RA3-6B2; 1:100), Gr1 (RB6-8C5; 1:600), Ter119 (Ter-119; 1:100) and CD127 (A7R34; 1:100). The cells were then washed and stained with mAbs to CD34 (RAM34; 1:10), Sca1 (D7; 1:100), CD117 (ACK2; 1:100) and SA-APCe780 for 60–90 minutes. For assessment of intracellular signaling pathways, the following antibodies from Cell Signaling Technologies were used: phosphorylated S6 (D572.2E; 1:200), IkBα (L35A5; 1:100) and phosphorylated STAT1 (58D6; 1:10). Data were acquired on a LSRII or FACSCanto (BD Biosciences) or cells sorted on a FACSAria and analyzed using FlowJo (TreeStar).

Buechler M.B., Akilesh H.M, & Hamerman J.A. (2016). Direct sensing of TLR7 ligands and Type I IFN by the common myeloid progenitor promotes mTOR/PI3K-dependent emergency myelopoiesis. Journal of immunology (Baltimore, Md. : 1950), 197(7), 2577-2582.

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9

Calcium Flux Measurement in Lineage-Depleted Bone Marrow

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BM was freshly isolated and lineage depleted with the MACS Lineage Depletion Kit (Miltenyi Biotech, San Diego, CA). Cells were cultured for 30min in complete medium supplemented with 1µM Indo-1 prepared as stock supplemented with Pluronic-F127 and incubated at 37 °C for 30min. Cells were washed and stained for surface markers for 15min, washed and allowed to rest in for 15min PBS in PBS with Ca2⁺. FACS tubes were run at 37 °C in the sample port of the LSRII flow cytometer equipped with a 355nM excitation laser. Events were collected for 40 seconds prior to incubation with 25uM ATP or 1uM SDF1 to induce calcium transients. The average ratio, R, of bound/free Indo-1 (405nm/485nm emission) before simulation was used to determine baseline values. Identical samples were equilibrated in 10mM EDTA PBS w/o Ca2⁺ to determine R_min or stimulated with 1uM ionomycin to determine R_max. The Indo-1 dissociation constant (K_d) was assumed to be 237nM at 37 °C based on previous studies.⁴⁰ The following equation was then used to relate Indo-1 intensity ratios to [Ca_i²⁺] levels;

[{Ca}^{2 +}] = K_{d} \cdot \frac{(R - R_{min})}{(R_{max} - R)}

Luchsinger L.L., de Almeida M.J., Corrigan D.J., Mumau M, & Snoeck H.W. (2016). Mitofusin 2 maintains hematopoietic stem cells with extensive lymphoid potential. Nature, 529(7587), 528-531.

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Macs lineage depletion kit

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Close spelling variants of this product

9 protocols using macs lineage depletion kit

Generation of Bone-Marrow Chimeras

Generation of Bone-Marrow Chimeras

Hematopoietic Stem Cell-Derived Dendritic Cells

Calcium Flux Measurement in Lineage-Depleted Bone Marrow

Gamma Radiation Effects on Bone Marrow Transplants

Generating Bone-Marrow Chimeras in NOD Mice

Generating Myeloid Progenitor Cells from Bone Marrow

Isolation and Characterization of Murine Hematopoietic Stem Cells

Calcium Flux Measurement in Lineage-Depleted Bone Marrow

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