Cd150 tc15 12f12.2

Manufactured by BioLegend

Sourced in United States

CD150 (TC15-12F12.2) is a mouse monoclonal antibody that recognizes the CD150 antigen. CD150 is a cell surface receptor that plays a role in lymphocyte activation and development.

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Lab products found in correlation

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Close spelling variants of this product

CD150 (24 citations) Anti-CD150 (TC15-12F12.2) (7 citations) TC15-12F12.2 (6 citations) Phycoerythrin-conjugated anti-CD150/SLAMf1 (clone TC15-12F12.2). (2 citations)

15 protocols using cd150 tc15 12f12.2

Immunophenotyping of Hematopoietic Cells

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Primary antibodies used for IHC and flow cytometry were as follows: c-Kit (2B8; eBioscience), CD16/32 (93; eBioscience), VE-cadherin (eBioscience), c-Kit (R&D Systems), Sca-1 (E13-161.7; BioLegend), CD48 (HM48-1; BioLegend), CD150 (TC15-12F12.2; BioLegend), IL-7Rα (SB/199; BioLegend), endoglin (MJ7/18; BioLegend), CD4 (L3T4; BD), CD8 (53-6.72; BD), B220 (RA3-6B2; BD), TER-119 (BD), Gr-1 (RB6-8C5; BD), CD34 (RAM34; BD), Mac-1 (M/70; BD), Flt-3 (A2F10.1; BD), CD41 (MWReg30; BD), CD45.2 (104; BD), CD45.1 (A20; BD), GPIbα (Xia.G5; Emfret), Clec2 (AbD Serotec), and Thpo (Bioss). Secondary antibodies for IHC were Alexa Fluor 488–conjugated IgGs (Molecular Probes) or Cy3/Cy5/DyLight549/DyLight649-conjugated IgGs (Jackson ImmunoResearch Laboratories, Inc.). IHC specimens were treated with DAPI (Molecular Probes) for nuclear staining. Stimulatory rabbit anti–mouse CLEC-2 antibody was a gift of K. Suzuki-Inoue.

Nakamura-Ishizu A., Takubo K., Kobayashi H., Suzuki-Inoue K, & Suda T. (2015). CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow. The Journal of Experimental Medicine, 212(12), 2133-2146.

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Isolating Hematopoietic Stem Cells from Bone Marrow

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Single cell suspensions from BM were prepared as described previously.²⁷ Red blood cells (RBC) were removed from BM cells using RBC lysis buffer (Sigma), and cells were stained with anti‐CD16/32 antibody (93, BioLegend) for blocking. Monoclonal antibodies (mAbs) used for this assay were the anti‐lineage cocktail (BD Biosciences), Sca‐1 (E13‐161.7; BioLegend), c‐Kit (2B8; BioLegend), Flk2/CD135 (A2F10; BioLegend), CD150 (TC15‐12F12.2; BioLegend) and CD48 (HM48‐1; BioLegend). All mAbs were purified and conjugated with either PerCP‐Cy5.5, PE‐Cy7, APC‐Cy7, APC, PE, and FITC. Flow cytometry was performed on a FACSCalibur instruments (BD Biosciences) and a FACS Aria instrument (BD Biosciences) was used for cell sorting. Data analysis was performed using FlowJo v10 software (FlowJo, LLC). Dead cells were excluded by propidium iodide (PI; Sigma) staining or by analyses using the two‐dimensional profile forward scatter vs side scatter.

Hsieh H., Jia W., Jin Z., Kidoya H, & Takakura N. (2020). High expression of PSF1 promotes drug resistance and cell cycle transit in leukemia cells. Cancer Science, 111(7), 2400-2412.

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Multiparametric Flow Cytometry Analysis

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All flow cytometric analyses and sorting were performed on a BD FACS Aria II cell sorter. Antibodies used were CD4 (RM4-5, BD Biosciences), CD8a (53-6.7, eBioscience), B220 (RA3-6B2, Biolegend), CD11b (M1/70, BD Biosciences), Ly6G/C (RB6-8C5, Biolegend), Ter119 (TER-119, Biolegend), Sca1 (D7, Biolegend), MPL (AMM2, Immuno-Biological Laboratories), CD41 (MWReg30, BD Biosciences), CD150 (TC15-12F12.2, Biolegend), cKit (2B8, Biolegend), CD34 (RAM34, eBioscience), IL7R (A7R34, eBioscience), Flt3 (A2F10, eBioscience), CD16/32 (93, eBioscience) and CD105 (MJ7/18, Biolegend). Streptavidin (eBioscience) was used to resolve biotinylated antibodies and propidium iodide was added prior to analysis to identify live cells.

O’Neill A., Chin D., Tan D., Majeed A.B., Nakamura-Ishizu A, & Suda T. (2020). Thrombopoietin maintains cell numbers of hematopoietic stem and progenitor cells with megakaryopoietic potential. Haematologica, 106(7), 1883-1891.

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Hematopoietic Progenitor Cell Enrichment

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Lin− cells were selected using EasySep™ Mouse Hematopoietic Progenitor Cell Enrichment Kit (Stem Cell Technologies, Grenoble, France) and incubated with the following antibodies conjugated with PE, PE-Cy5, PE-Cy7, fluorescein isothiocyanate (FITC), allophycocyanin (APC) or APCeFluor®780: Sca1 (D7), cKit (2B8), CD48 (HM48-1), CD127 (A7R34), and CD150 (TC15-12F12.2; BioLegend, San Diego, USA). All reagents were purchased from Affymetrix (High Wycombe, UK), unless otherwise stated. Flow cytometry acquisition and sorting was performed using MoFlo XDP (Beckman Coulter, High Wycombe, UK).

Verbiest T., Finnon R., Brown N., Cruz-Garcia L., Finnon P., O’Brien G., Ross E., Bouffler S., Scudamore C.L, & Badie C. (2018). Tracking preleukemic cells in vivo to reveal the sequence of molecular events in radiation leukemogenesis. Leukemia, 32(6), 1435-1444.

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Comprehensive Lineage Analysis of Hematopoietic Cells

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For lineage analysis of peripheral blood, bone marrow, and spleen, flow cytometric analyses were performed as previously described (4 (link)). Myeloid progenitors in bone marrow and spleen were analyzed as previously described (8 (link)). HSCs in bone marrow and spleen were analyzed as described in (23 (link)). Because hind limb bone marrow represents ~25% of total bone marrow, the number of HSCs in total bone marrow is calculated as 4 X the number of HSCs in hind limb bone marrow. The stained cells were analyzed on a FACS Calibur (BD Biosciences) or a MACSQuant Analyzer (Miltenyi Biotec Inc.). Antibodies specific for the following surface antigens were purchased from eBioscience: CD45.2 (104), B220 (RA3-6B2), CD19 (eBio1D3), Thy1.2 (53-2.1), Mac-1 (M1/70), Gr-1 (RB6-8C5), CD4 (GK1.5), CD8 (53-6.7), CD3 (145-2C11), IgM (II/41), IL7Rα (A7R34), Sca-1 (D7), TER119(TER-119), CD34 (RAM34), cKit (2B8). FcγRII/III (2.4G2) was purchased from BD Biosciences. CD150 (TC15-12F12.2) was purchased from Biolegend.
Additional methods are described in Supplemental Methods.

Chang Y.I., You X., Kong G., Ranheim E.A., Wang J., Du J., Liu Y., Zhou Y., Ryu M.J, & Zhang J. (2015). Loss of Dnmt3a and endogenous KrasG12D/+ cooperate to regulate hematopoietic stem and progenitor cell functions in leukemogenesis. Leukemia, 29(9), 1847-1856.

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Multiparameter Flow Cytometry Immunophenotyping

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The following monoclonal antibodies (Abs) were used for flow cytometry and cell sorting: anti-c-Kit (2B8, BD Biosciences), -Sca-1 (E13-161.7/D7, BioLegend), -CD140a (APA5, BD Biosciences), -CD51 (RMV-7, BIO-RAD), -CD4 (RM4-5, BioLegend), -CD8a (53-6.7, BioLegend), -B220 (RA3-6B2, BioLegend/WAKO), -CD31 (390, BioLegend), -TER-119 (TER-119, BioLegend/BD Biosciences), -Gr-1 (RB6-8C5, BD Biosciences), -Mac-1 (M1/70, BioLegend), -CD48 (HM48-1, BD Biosciences), -CD150 (TC15-12F12.2, BioLegend), -CD19 (6D5, BioLegend), -IgM (MHM-88, BioLegend), -CD43 (S11, BioLegend), -CD45.1 (A20, BD Biosciences), -CD45.2 (104, BD Biosciences), -CD45 (30-F11, BioLegend), -Flt3 (A2F10.1, BD Biosciences), -IL7Rɑ (A7R34, TONBO biosciences), -FcγR (2.4G2, TONBO biosciences), -53BP1 (Novus Biologicals). A biotinylated CD34 (RAM34, Thermo Fisher Scientific) and a fluorochrome labelled streptavidin (BD Biosciences/BioLegend) were used.

Nakashima K., Kunisaki Y., Hosokawa K., Gotoh K., Yao H., Yuta R., Semba Y., Nogami J., Kikushige Y., Stumpf P.S., MacArthur B.D., Kang D., Akashi K., Ohga S, & Arai F. (2023). POT1a deficiency in mesenchymal niches perturbs B-lymphopoiesis. Communications Biology, 6, 996.

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Quantifying Mitochondrial Membrane Potential in HSCs

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Mitochondrial membrane potential within HSCs was quantified by flow cytometry as follows^{27 (link)}. Briefly, WBM cells from femurs and tibiae were depleted of lineage committed hematopoietic progenitors and surface stained for 30 minutes at 4⁰C with antibodies raised against SCA1 (D7; Biolegend), cKIT (2B8; Biolegend), CD150 (TC15-12F12.2; Biolegend), and CD48 (HM48-1; Biolegend). Following surface staining, cells were washed with MACS buffer and re-suspended in micro-centrifuge tubes containing 0.5 mL DMEM with TMRE (100 nM, ThermoFisher Scientific T669) and Verapamil (50 µM, Sigma-Aldrich V4629). Cells were incubated in a 37 °C CO₂ incubator for 25 minutes with caps open. Following incubation, cells were washed twice with ice-cold MACS buffer. Washed cells were re-suspended in ice-cold MACS buffer and maintained at 4 °C under low-light conditions until flow cytometry acquisition. Cells incubated in DMEM without TMRE served as gating controls. TMRE sensitivity was confirmed by collapse of TMRE intensity to background levels after mitochondrial uncoupling induced by 20 μM FCCP.

Ramalingam P., Gutkin M.C., Poulos M.G., Tillery T., Doughty C., Winiarski A., Freire A.G., Rafii S., Redmond D, & Butler J.M. (2023). Restoring bone marrow niche function rejuvenates aged hematopoietic stem cells by reactivating the DNA Damage Response. Nature Communications, 14, 2018.

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Isolation and Characterization of Mouse Hematopoietic Stem and Progenitor Cells

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Mouse BM cells were harvested and stained as previously described. Briefly, antibodies used in this study include a lineage cocktail containing antibodies against Ter-119 (clone Ter-119), B220 (RA3-6B2), CD3e (145-2C11), CD4 (GK1.5), CD8 (53–6.7), Gr-1 (RB6-8C5), and Mac-1 (M1/70), conjugated with PE-Cy5 (eBiosciences). Additional antibodies used to identify HSPCs included CD16/32 (93) in Alexa eFluor 700, IL7Ra (A7R34) in PE-Cy5, CD45.1 (A20) in PE-Cy7, c-Kit (2B8) in allophycocyanin-eFluor 780, Gr-1 (RB6-8C5) in PE, CD45.2 (104) in Alexa eFluor 700, CD34 (RAM34) in fluorescein isothiocyanate, Flk2/Flt3 (A2F10) in PE (all from eBiosciences), as well as Sca-1 (E13-161.7) in Pacific Blue and CD150 (TC15-12F12.2) in PE (both from BioLegend). After staining, cells were analyzed and sorted using a FACSAria II (Becton Dickinson). All cell populations were double sorted, and a purity of >90% was routinely achieved. Mouse CD97 v2 Alexa Fluor 488–conjugated antibody (R&D Systems) and mouse anti-human CD97 allophycocyanin conjugate (clone MEM-180; Life Technologies) were used to stain mouse and human CD97, respectively. Flow cytometry data were analyzed using Flowjo software (TreeStar).

Martin G.H., Roy N., Chakraborty S., Desrichard A., Chung S.S., Woolthuis C.M., Hu W., Berezniuk I., Garrett-Bakelman F.E., Hamann J., Devlin S.M., Chan T.A, & Park C.Y. (2019). CD97 is a critical regulator of acute myeloid leukemia stem cell function. The Journal of Experimental Medicine, 216(10), 2362-2377.

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Flow Cytometry Antibody Panel for Murine Hematopoietic Cells

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The following antibodies were used: rat mAbs against 1/200 cKit (2B8) (Biolegend Cat. no. 105814), 1/100 Sca1 (D7) (Biolegend Cat. no. 108112), 1/100 CD150 (TC-15-12F12.2), 1/25 CD34 (RAM34), 1/100 CD48 (HM48-1) (Biolegend Cat. no. 103418), 1/200 CD45.2 (104) (Biolegend Cat. no. 109820), 1/200 CD45.1 (A20) (Biolegend Cat. no. 110706), 1/1,000 Gr1 (RB6-8C5) (Biolegend Cat. no. 108412), 1/750 F4/80 (BM8), 1/500 CD19 (6D5) (Biolegend Cat. no. 115507), 1/200 CD3 (17A2) (Biolegend Cat. no. 100206), 1/500 CD16/CD32 (2.4G2) (BD Cat. no. 553141). The antibodies were purchased from Biolegend, eBiosciences and BD (Becton, Dickinson and Company). A mixture of biotinylated mAbs against CD3, CD11b, CD45R/B220, Ly-6G, Ly-6C and TER-119 was used as lineage cocktail (BD).

Vannini N., Girotra M., Naveiras O., Nikitin G., Campos V., Giger S., Roch A., Auwerx J, & Lutolf M.P. (2016). Specification of haematopoietic stem cell fate via modulation of mitochondrial activity. Nature Communications, 7, 13125.

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Murine Hematopoietic Cell Immunophenotyping

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Directly conjugated or biotin-conjugated antibodies specific for the following surface antigens were purchased from eBioscience: CD3 (145–2C11), CD4 (RM4–5), CD8 (53–6.7), B220 (RA3–6B2), TER119 (TER-119), Gr-1 (RB6–8C5), IgM (eB121–15F9), CD19 (eBio1D3), IL-7Ra (A7R34), CD45.2 (104), CD45.1 (A20), Sca-1 (D7), c-Kit (2B8), Mac1 (M1/70), Gr1 (RB6–8C5), Thy1.2 (53–2.1), and CD19 (eBio1D3). CD48 (103427) and CD150 (TC15–12F12.2) were purchased from Biolegend. Acquisitions were performed on a LSR II or LSR II Fortessa (BD Biosciences). Data were analyzed using BD FlowJo v10.0.7 software. For cell sorting, Lin⁻c-Kit⁺Sca-1⁺CD150²²¹²HSCs or Lin⁻GFP⁺ were sorted from freshly prepared single BM suspension by the University of Wisconsin-Madison Carbon Cancer Center Flow Cytometry and Cell Sorting Facility.

Lu Z., Hong C.C., Kong G., Assumpção A.L., Ong I.M., Bresnick E.H., Zhang J, & Pan X. (2018). Polycomb Group Protein YY1 Is an Essential Regulator of Hematopoietic Stem Cell Quiescence. Cell reports, 22(6), 1545-1559.

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