Nk1.1 pk136

Manufactured by Thermo Fisher Scientific

Sourced in United States

NK1.1 (PK136) is a mouse monoclonal antibody that recognizes the NK1.1 antigen, a type II transmembrane glycoprotein expressed on natural killer cells and a subset of T cells in mice. The NK1.1 antibody is commonly used as a marker for the identification and isolation of natural killer cells in flow cytometry and other immunological applications.

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24 protocols using nk1.1 pk136

Multiparametric Immune Cell Profiling

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Peripheral blood was collected retro-orbitally from isoflurane-anesthetized mice. The blood was treated with ACK lysis buffer for 5 min to remove RBCs, leaving the peripheral blood leukocytes (PBLs). To determine expression of cell surface molecules, we incubated PBLs with mAb at 4°C for 20–30 min, and cells were subsequently fixed for 10 min using Cytofix Solution (BD Biosciences). The following mAb clones were used to stain processed samples: CD11a (M17/4; eBioscience), TLR4 (SA15-21; BioLegend), CD3ε (145-2C11; BioLegend), NK1.1 (PK136; eBioscience), NKp46 (29A1.4; BioLegend), F4/80 (BM8; BioLegend), TLR2 (CB225; BD Bioscience), CD19 (6D5; BioLegend), CD11c (HL3; BD Biosciences), CD4 (GK1.5; BioLegend), Ly6G (1A8; BioLegend), CD127 (A7R34; BioLegend), Ly6C (HK1.4; BioLegend), and CD8α (53-6.7; BioLegend). Flow cytometry data were acquired on a Cytek Aurora (Cytek, Bethesda, MD) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Berton R.R., Jensen I.J., Harty J.T., Griffith T.S, & Badovinac V.P. (2022). Inflammation Controls Susceptibility of Immune-Experienced Mice to Sepsis. ImmunoHorizons, 6(7), 528-542.

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Multiparameter Flow Cytometry Analysis

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Lymphocyte surface markers were stained with antibodies against CD19 (6D5), H57 (H57-597), CD8 (53-6.7), CD44 (IM7), CD122 (TM-b1), and NK1.1 (PK136) (eBioscience and BioLegend). C2C12 cells were stained with biotin-conjugated antibodies against IL-15 (Cat. No. 500-P173Bt, PeproTech), IL-15Rα (Cat. No. BAF551, R&D), IL-15Rβ (CD122, clone: TM-b1, eBioscience), and γ_c (CD132, Cat. No. 554470, BD Biosciences) then incubated with APC-conjugated streptavidin (BD Biosciences). CD8⁺ T cells were stained with fixable viable dye eFluor506 (eBioscience) then intracellularly stained with antibodies against IFN-γ (XMG1.2, eBioscience) and granzyme B (NGZB, eBioscience). All data were acquired on LSRII (BD Biosciences) and analyzed by the FlowJo (Tree Star).

Huang P.L., Hou M.S., Wang S.W., Chang C.L., Liou Y.H, & Liao N.S. (2015). Skeletal muscle interleukin 15 promotes CD8+ T-cell function and autoimmune myositis. Skeletal Muscle, 5, 33.

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Isolation of Hematopoietic Stem Cells

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HSCs (Tie2⁺CD150⁺CD48^low/−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)}.

Turcotte R., Alt C., Runnels J.M., Ito K., Wu J.W., Zaher W., Mortensen L.J., Silberstein L., Côté D.C., Kung A.L., Ito K, & Lin C.P. (2017). Image-guided transplantation of single cells in the bone marrow of live animals. Scientific Reports, 7, 3875.

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Comprehensive Immunophenotyping of Murine Immune Cells

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The antibodies with following specificities were used; CD4 (GK1.5, Tonbo), CD8 (53.67, Tonbo), CD11c (HL3, BD), CD11b (M1/70, eBioscience), TCRβ (H57-597, BD), NK1.1 (PK136, eBioscience), γδTCR (GL3, Biolegend), CD44 (IM7, eBioscience), CD122 (TMβ1, BD), CD62L (MEL-14, eBioscience), IL-15Rα (DNT15Rα, eBioscience), IL-2Rα (3C7, Biolegend), γc (4G3, BD), IL-17 (eBio17B4, eBioscience), IFNγ (XMG1.2, Biolegend), pSTAT5 (clone 47, BD), Foxp3 (MF23, BD). Fluorochrome-conjugated CD1d tetramers loaded with PBS-57 were obtained from the NIH tetramer facility (Emory University, Atlanta, GA).

Waickman A.T., Ligons D.L., Hwang S., Park J.Y., Lazarevic V., Sato N., Hong C, & Park J.H. (2017). CD4 effector T cell differentiation is controlled by IL-15 that is expressed and presented in trans. Cytokine, 99, 266-274.

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Analyzing Mouse Hematopoietic Cell Subsets

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Bone marrow (BM) cells isolated from the femur and tibia were prepared as described previously (Lei et al., 2018 (link)). All collected cells were treated with ACK (red blood cell lysis) buffer before analysis. To analyze hematopoietic cells, monoclonal antibodies from BD Biosciences (San Jose, CA, United States), eBioscience (San Diego, CA, United States), and BioLegend (San Diego, CA, United States) recognizing the following surface markers were used: Sca-1 (D7), c-Kit (2B8), CD150 (mShad150), CD48 (HM48-1), CD127 (A7R34), CD16/32 (93), Gr-1 (RB6-8C5), Mac-1 (M1/70), B220 (RA3-6B2), CD3e (145-2C11), CD45.1 (A20), CD45.2 (104), Ki67 (7B11), and streptavidin. The mouse lineage cocktail contained anti-CD3, Mac-1, Gr-1, B220, Ter-119 (TER-119), and NK1.1 (PK136) antibodies (eBioscience). Cell analysis was performed using a FACS LSR Fortessa (BD Biosciences) and cell sorting was performed using a FACS AriaIII (BD Biosciences). Data were analyzed using FlowJo10.0 software (TreeStar, Ashland, OR, United States).

Liu Y., Chen Y., Deng X, & Zhou J. (2020). ATF3 Prevents Stress-Induced Hematopoietic Stem Cell Exhaustion. Frontiers in Cell and Developmental Biology, 8, 585771.

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Bone Marrow Reconstitution Assay

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Briefly, bone marrow cells were collected and labeled with anti-Thy1.2 (30-H12, Ebioscience) and NK1.1 (PK136, Ebioscience). Cells were washed and resuspended with rabbit complement (Cedarlane Laboratories). After complement lysis, cells were washed with media containing 10% fetal calf serum and wild-type cells and CD25 deficient cells were mixed in equal portions. Recipient mice were lethally irradiated (1000 rads) and injected with 5×10⁶ total bone marrow cells and given antibiotic treated water for 6 weeks.

Hondowicz B.D., Kim K.S., Ruterbusch M.J., Keitany G.J, & Pepper M. (2017). IL-2 is required for the generation of viral-specific CD4+ Th1 Trms cells and B cells are essential for maintenance in the lung. European journal of immunology, 48(1), 80-86.

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Immunophenotyping of Murine Bone Marrow

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Total BM cells were flushed and harvested from both hind legs. Single-cell suspensions from the spleen were obtained by enzymatic digestion as previously described.^{27 (link)} Fluorochrome-conjugated anti-mouse B220 (RA3-6B2), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (eBio1D3), CD16/32 (93), CD34 (RAM34), CD45.1 (A20), CD45.2 (104), CD48 (HM48-1), CD117/c-Kit (2B8), CD135/Flt3 (A2F10), CD150 (TC15-12F12.2), F4/80 (BM8), CD127/IL-7Rα (A7R34), Ki-67 (SolA15), Ly-6G (RB6-8C5), Sca-1 (D7) and Siglec-H (eBio440C) antibodies were used for surface immunophenotyping (eBioscience; San Diego, CA, USA). Lineage-positive cells in the BM were excluded using the following biotinylated antibodies, followed by treatment with APC-Cy7-conjugated streptavidin: B220, CD3, CD4, CD8, CD11b, CD11c, CD19, Gr-1, NK1.1 (PK136) and Ter119 (TER-119) (all from eBioscience). Cell death and apoptosis were analyzed using an Annexin V/propidium iodide (PI) staining kit (eBioscience). Stained cells were acquired using a FACSVerse or LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA). All flow cytometry data were analyzed with the FlowJo software (Treestar, Ashland, OR, USA).

Kim T.G., Kim S., Jung S., Kim M., Yang B., Lee M.G, & Kim H.P. (2017). CCCTC-binding factor is essential to the maintenance and quiescence of hematopoietic stem cells in mice. Experimental & Molecular Medicine, 49(8), e371-.

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Comprehensive Thymic γδ T Cell Phenotyping

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Abs against Thy1.2 (53–2.1), CD24 (M1/69), CD25 (PC61), CD44 (IM7), TCR-δ (GL3), TCR-β (H57-597), CD4 (GK1.5), CD8α (53–6.7), CD27 (LG.7F9), CD45RB (C363-16A), CD73 (Ty/11.8), Vγ2 (UC3-10A6), Vγ3 (536), and NK1.1 (PK136) were purchased from eBioscience, BD, or BioLegend. Anti-Vγ1.1 antibody was provided by R. O’Brien (National Jewish Medical Center, Denver, CO). Propidium iodide gating was included for dead cell exclusion. Data were collected on either a LSRII or FACSVantage SE (BD) and analyzed using FlowJo Software (Tree Star). For isolation of thymic γδ T cell populations, thymocytes were harvested from 6–7-wk-old C57BL/6 mice, depleted with anti-CD4 and anti-CD8 magnetic beads (Miltenyi Biotec), and isolated by cell sorting. A dump gate (NK1.1, B220, Ter11, Gr-1, and CD11b) was included. Intracellular TCR chains were detected by blocking surface TCR using unlabeled Ab, fixing for 10 min with 1% paraformaldehyde at room temperature, permeabilization with saponin and NP-40 detergents, and then staining with fluorochrome-conjugated anti–TCR-β and TCR-δ Ab.

Coffey F., Lee S.Y., Buus T.B., Lauritsen J.P., Wong G.W., Joachims M.L., Thompson L.F., Zúñiga-Pflücker J.C., Kappes D.J, & Wiest D.L. (2014). The TCR ligand-inducible expression of CD73 marks γδ lineage commitment and a metastable intermediate in effector specification. The Journal of Experimental Medicine, 211(2), 329-343.

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Multiparametric Immune Cell Analysis

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MAbs directed against mouse CD3 (145–2C11), CD11b (M1/70), CD11c (N418), F4/80 (BM8), B220 (RA3-6B2) and NK1.1 (PK136) were from eBioscience or BD Pharmingen. Staining buffer consisted of PBS containing 2% BSA plus 0.1% sodium azide.

Graham K.L., Zhang J.V., Lewén S., Burke T.M., Dang T., Zoudilova M., Sobel R.A., Butcher E.C, & Zabel B.A. (2014). A Novel CMKLR1 Small Molecule Antagonist Suppresses CNS Autoimmune Inflammatory Disease. PLoS ONE, 9(12), e112925.

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Multicolor Flow Cytometry Immunophenotyping

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Flow cytometry data were acquired on a FACSCanto (BD Biosciences, San Diego, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR). To determine expression of cell surface proteins, mAb were incubated at 4°C for 20–30 min and cells were fixed using Cytofix/Cytoperm Solution (BD Biosciences) and, in some instances, followed by incubation with mAb for an additional 20–30 min to detect intracellular proteins. The following mAb clones were used to stain murine samples: NK1.1 (PK136; eBioscience), CD3 (17A2; eBioscience), NKp46 (29A1.4; eBioscience), IFNγ (XMG1.2; eBioscience), Thy1.1 (HIS51; eBioscience), CD49b (DX5; Biolegend), CD122 (TM-b1; eBioscience), and CD19 (MB19–1; eBioscience). For cytokine staining following in vitro stimulation BFA (BD Biosciences) was added during the last four hours of stimulation. The following mAb clones were used staining of patient samples: CD45 (HI30; Tonbo), CD3 (OKT3; Tonbo), CD19 (HIB19; Tonbo), CD56 (MY31; Tonbo), and IL-10 (JES5–2A5; Tonbo).

Jensen I.J., McGonagill P.W., Butler N.S., Harty J.T., Griffith T.S, & Badovinac V.P. (2021). NK cell-derived IL-10 supports host survival during sepsis. Journal of immunology (Baltimore, Md. : 1950), 206(6), 1171-1180.

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