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.
Cd48 hm48 1
Manufactured by BioLegend
Sourced in United States
CD48 (HM48-1) is a lab equipment product from BioLegend that is used to detect the presence and expression levels of the CD48 cell surface antigen. CD48 is a glycosylphosphatidylinositol (GPI)-anchored protein that functions as a ligand for the CD2 receptor, playing a role in cell-cell adhesion and activation.
Automatically generated - may contain errors
Lab products found in correlation
Cd150 tc15 12f12.2, by BioLegend (6 mentions)
C kit 2b8, by BioLegend (4 mentions)
Sca1 d7, by BioLegend (3 mentions)
Cd16 32, by Thermo Fisher Scientific (2 mentions)
Ter119, by BD (2 mentions)
C kit 2b8, by Thermo Fisher Scientific (2 mentions)
Sca 1 e13 161.7, by BioLegend (2 mentions)
Cd4 l3t4, by BD (2 mentions)
Cd34 ram34, by BD (2 mentions)
Cd41 mwreg30, by BD (2 mentions)
Cd45.1 a20, by BD (2 mentions)
B220 ra3 6b2, by BD (2 mentions)
Cd45.1 a20, by BioLegend (2 mentions)
Gr 1 rb6 8c5, by BioLegend (2 mentions)
Cd3e 145 2c11, by BioLegend (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ve cadherin, by Thermo Fisher Scientific (1 mentions)
Gr 1 rb6 8c5, by BD (1 mentions)
Cd8 53 6.72, by BD (1 mentions)
Mac 1 m 70, by BD (1 mentions)
10 protocols using cd48 hm48 1
1
Immunophenotyping of Hematopoietic Cells
2
Isolation of Hematopoietic Stem Cells
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HSCs (Tie2+CD150+CD48low/−CD135− LKS) were harvested from the Tie2 GFP mouse and sorted as described in Ito, K et al.18 (link), using monoclonal antibodies specific for the following: CD41 (eBioMWRag30), CD135 (Avas12a1), CD34 (RAM34), c-Kit (2B8), Sca-1 (E13-161.7), CD3e (145-2C11), CD4 (L3T4), CD8 (53-6.72), B220 (RA3-6B2), TER-119 (TER-119), Gr-1 (RB6-8C5), CD11b (M1/70), IgM (II/41), CD19 (eBio1D3), F4/80 (BM8), CD25 (PC61), CD44 (IM7), CD71 (R17217), CD127 (A7R34), CD45.2 or Ly5.2, (104) CD45.1 or Ly5.1 (A20) and NK-1.1 (PK136); all were from eBioscience. Anti-CD150 (TC15-12F12.2) and CD48 (HM48-1) antibodies were from BioLegend. We used a mixture of monoclonal antibodies against CD4, CD8, CD3e, B220, TER-119, CD11b, Gr-1, IgM, CD19, CD127, and NK-1.1 as a lineage marker (Lineage)18 (link).
3
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.
4
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).
5
Multiparameter Flow Cytometry Analysis
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The following mAbs were used in this study: rat mAbs against CD16/32 (93; eBioscience), c-Kit (2B8; eBioscience and Tonbo Biosciences), Sca-1 (E13-161.7; BioLegend), CD4 (L3T4; BD Biosciences), CD8a (53-6.72; BD Biosciences), B220 (RA3-6B2; BD Biosciences), TER-119 (TER119; Tonbo Biosciences), Gr-1 (RB6-8C5; BD Biosciences), CD34 (RAM34; eBioscience), Mac-1 (M1/70; BD Biosciences and eBioscience), Flt3 (A2F10.1; BioLegend), CD45.2 (104; BD Biosciences), CD45.1 (A20; BD Biosciences), IL7Rα (SB/119; BioLegend), CD41 (MWReg30; BD Biosciences), CD48 (HM48-1; BioLegend), CD150 (TC15-12F12.2; BioLegend), and CD45 (30-F11; BioLegend and eBioscience and BD Biosciences). A mixture of mAbs against CD4, CD8, B220, TER-119, Mac-1, and Gr-1 was used as a lineage marker (lineage). A mouse anti–Ki67-Alexa Fluor 555 antibody (B56; BD Biosciences) was used to assess the cell cycle, and mouse antiphosphorylated-p38MAPK–phenylephrine (PE) antibody (36/p38; BD Biosciences) and mouse anti-IgG1–PE antibody (BD Biosciences) were used to detect phosphorylated p38MAPK by intracellular flow cytometry. Annexin V–PE (556422; BD Bioscience) was used to analyze apoptosis. For immunocytochemistry, an anti-H2AX (pS139) antibody (N1-431; BD Bioscience), a rabbit anti-53BP1 polyclonal antibody (NB100-304; Novus Biologicals), and Alexa Fluor 555–conjugated anti-rabbit IgG (A-21428; Invitrogen) were used.
6
Multiparameter Phenotyping of Hematopoietic Cells
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Single-cell suspensions from spleen, blood, BM, or BALF were washed in PBS containing 5 mM ethylenediaminetetraacetic acid and 0.5% BSA. Cells were incubated for 15 min with Fc receptor block (clone 2.4G2; BD) before antibody staining. The following antibodies were used for FACS staining and sorting: biotinylated antibodies for lineage-negative staining-biotin lineage staining cocktail includes CD3, B220, GR-1, CD11b, and Ter119, as well as NK1.1, CD11c, and DX-5 (BD), were added to the staining cocktail; CD45.2 (104), cKit (2B8), and CD34 (Ram34) were purchased from BD; CD16/32 (93), Sca-1 (D7), and CD135 (A2F10) were purchased from eBioscience; CD45.1 (A20), CD48 (HM48-1), and CD150 (TC15-12F12.2) were purchased from BioLegend. Aqua Fluorescent reactive dye (Life Technologies) was used to discriminate viable from dead cells. Intracellular BrdU and 7AAD for cell cycle analysis using the FITC BrdU Flow kit (BD) was performed per the manufacturer’s protocol. Flow cytometry data was analyzed using FlowJo software (Tree Star).
7
Monoclonal Antibodies for Cell Identification
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The monoclonal antibodies used in this study targeted the following proteins: CRLR (H-42; Santa Cruz, Dallas, TX, USA), RAMP-1 (EPR10867; abcam, Cambridge, UK), Gr-1 (RB6-8C5; BioLegend, San Diego, CA, USA), Mac-1 (M1/70; BioLegend), B220 (RA3-6B2; BioLegend), CD3e (145-2C11; Biolegend), CD2 (RM2-5; TONBO, Japan), CD8a (53–6.72; TONBO), TER-119 (TER-119; TONBO), CD127 (A7R34; TONBO), c-Kit (2B8; BioLegend), Sca-1 (D7; BioLegend), CD34 (RAM34; eBioscience, Santa Clara, CA, USA), CD16/32 (93; eBioscience), CD48 (HM48-1; BioLegend), CD150 (TC15–12F12.2; BioLegend), CD45.2 (104; eBioscience) and CD45.1 (A20; eBioscience). A mixture of mAbs against CD2, CD3e, CD8a, B220, TER-119, CD127, Mac-1 and Gr-1 served as lineage markers (Lineage). Mouse anti-BrdU-Alexa Fluor 647 antibody (3D4; BD Biosciences) was used to detect intracellular BrdU.
8
Murine Hematopoietic Stem Cell Immunophenotyping
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All analyses were done on FACS Aria II and Canto (BD Bioscience). The antibodies used for staining are mKi67 (1:100/Cat. 11-5698−82), CD117 (2B8) (1:600/Cat. 47-1171−80), Sca-1 (D7) (1:100/Cat. 15-5981-81), Ter119 (Ter119) (1:200/Cat. 15-5921-81), CD41 (MWReg30) (1:400 (fluorescein isothiocyanate (FITC))–1:800 (allophycocyanin)/Cat. 11-0411-82), CD105 (MJ7/18) (1:200/Cat. 12-1051-82), CD16/32 (93) (1:50/Cat. 56-0161), CD11b (M1/70) (1:1200/Cat. 12-0112-81), Gr-1 (RB6-8C5) (1:800/Cat. 48-5931), CD3e (1:200/Cat. 17-0031-82), and CD45R (1:400/Cat. 13-0452-82) all from eBioscience. CD48 (HM48-1) (1:300/Cat. 103411) and CD150 (TC15-12F1) (1:50/Cat. 115914) are from BioLegend.
9
Cryosectioning and Immunofluorescence Staining
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Tissue was embedded in Tissue-Tek OCTTM compound (10.24% polyvinyl alcohol, 4.26% polyethylene glycol, 85.50% non-reactive ingredient) and sections of 10μm thickness prepared using a Reichert-Jung 2800 Frigocut cryostat (Reichert-Jung: Depew, New York, USA). Sections were acetone-fixed for 15 minutes at 4°C and air dried for an hour before storage at -80°C. Frozen sections were left to thaw at room temperature before staining. Tissue sections were first blocked with 10% heat inactivated fetal calf serum in PBS for 45 minutes at room temperature and then stained with fluorochrome-labelled antibody for 45 minutes at room temperature. Slides were washed thrice in PBS for 5 minutes. Stained sections were examined using a Leica TCS SP5 Confocal microscope (Leica Microsystems: North Ryde, NSW, Australia). Nuclear DNA was stained with DAPI (Sigma-Aldrich, St Louis, MO, USA). Antibodies used were specific for CD11b (M1/70), CD11c (HL3), H-2Kk (36-7-5), CD150 (TC15-12F12.2), CD41 (MWReg30) and CD48 (HM48-1) all purchased from Biolegend (San Diego, CA, USA).
10
Immunophenotypic Characterization of Murine Hematopoietic Stem and Progenitor Cells
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Bone marrow cells were isolated from femurs and tibias and treated with ACK red cell lysis buffer. Cells were stained with combinations of the following antibodies against cell-surface markers to identify indicated cell types (all antibodies are from Becton Dickinson unless indicated; clone indicated in parentheses): CD11b (MI/70), Gr1 (TB6-8C5; Biolegend), Ter-119 (Ter119), CD71 (C2), B220 (RA3-6B2), CD3e (145-2C11), Sca-1 (D7; Biolegend), c-KIT (2B8), CD34 (RAM34), FLT3 (A2F10; Biolegend), CD150 (TC15-12F12.2; Biolegend), CD48 (HM48-1; Biolegend), CD45.1 (A20), CD45.2 (104). Bone marrow and LinKit donor cells were isolated from transplanted mice using CD45.1-/CD45.2+ gating prior to selection for immunophenotypically defined populations. The following phenotypes were used to define stem and progenitor populations for sorting and Dnmt3a flow cytometry: Lineage (Lin): CD11b+, Gr1+, Ter119+, CD71+, B220+, CD3e+; LSK: Lin-, Sca-1+, c-KIT+; GMP: Lin-, Sca-1+, c-KIT+, CD34+, FLT3+; CMP: Lin-, Sca-1+, c-KIT+, CD34+, FLT3-; MEP: Lin-, Sca-1+, c-KIT+, CD34-, FLT3-. Sorting was performed on a modified Sony Synergy SY3200 (Sony Biotechnology, San Jose, CA) updated to 24 parameters. Winlist version 8 software was used for data acquisition and analysis (Verity Software House, Topsham, ME).
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