Nk1.1 clone pk136

Manufactured by BioLegend

Sourced in United States

NK1.1 clone PK136 is a mouse monoclonal antibody that recognizes the NK1.1 antigen. The NK1.1 antigen is expressed on natural killer (NK) cells and a subset of T cells in certain mouse strains. This antibody can be used to identify and characterize NK cells in various mouse models and applications.

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Lab products found in correlation

Cd19 clone 6d5, by BioLegend (5 mentions) Cd11b clone m1 70, by BioLegend (5 mentions) Cd4 clone rm4 5, by BioLegend (3 mentions) Cd8 clone 53 6, by BioLegend (3 mentions) Cd11c clone n418, by BioLegend (3 mentions) Cd3ε clone 145 2c11, by BioLegend (3 mentions) Ter119 clone ter 119, by BioLegend (3 mentions) Cd4 clone gk1, by BioLegend (3 mentions) Cd3 clone 145 2c11, by BioLegend (3 mentions) Cd3 clone 17a2, by BioLegend (2 mentions) Ly6g clone 1a8, by BioLegend (2 mentions) Ly6c clone hk1, by BioLegend (2 mentions) F4 80 clone bm8, by BioLegend (2 mentions) Cd45 clone 30 f11, by BioLegend (2 mentions) Cd11c clone n418, by Thermo Fisher Scientific (2 mentions) Pd l1 clone 10f 9g2, by BioLegend (2 mentions) Gr 1 clone rb6 8c5, by BioLegend (2 mentions) Cd19 clone 1d3, by BD (2 mentions) Lsrfortessa flow cytometer, by BD (2 mentions) Fortessa flow cytometer, by BD (2 mentions)

Close spelling variants of this product

NK1.1 (PK136) (26 citations) Anti-NK1.1 (clone PK136, (9 citations) PE/Cy7-anti-NK1.1 mAb (clone PK136, (2 citations) NK1.1-APC antibody (clone PK136, (1 citations)

15 protocols using nk1.1 clone pk136

Multiparameter Flow Cytometry Panel

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CD3 clone 17A2 (BioLegend 100237 and 100244), CD4 clone RM4–5 (BioLegend 100545 and 100510), CD4 clone GK13 (BioLegend 100403), CD8 clone 53–6.7 (BioLegend 100734), CD11b clone M1/70 (BioLegend 101257), CD11c clone N418 (BioLegend 117339 and 117338), CD19 clone 6D5 (BioLegend 115522), CD24 clone M1/69 (BioLegend 101822), CD45 clone 30-F11 (BioLegend 103139, 103132, and 103114; eBioscience 56–0451-82), CD45R clone RA3–6B2 (BioLegend 103247, 103246, and 103226), CD69 clone H1.2F3 (eBioscience 25–0691-81), CD90.2 clone 30-H12 (BioLegend 105331), CD103 clone 2E7 (BioLegend 121406 and 121414), F4/80 clone BM8 (BioLegend 123108), Flt3L (R&D Systems AF427), Ly6C clone HK1.4 (BioLegend 128037), Ly6G clone 1A8 (BioLegend 127645), MHC-II clone M5/114.15.2 (BioLegend 707631), NK1.1 clone PK136 (BioLegend 108707, 108720, and 108749), Streptavidin-Brilliant Violet 650 (BioLegend 405231), Streptavidin-APC (eBioscience 17–4317-82). Depleting antibodies: NK1.1 clone PK136 (BioXCell BE0036), and IgG2a isotype control (BioXCell BE0085).

Barry K.C., Hsu J., Broz M.L., Cueto F.J., Binnewies M., Combes A.J., Nelson A.E., Loo K., Kumar R., Rosenblum M.D., Alvarado M.D., Wolf D.M., Bogunovic D., Bhardwaj N., Daud A.I., Ha P.K., Ryan W.R., Pollack J.L., Samad B., Asthana S., Chan V, & Krummel M.F. (2018). A Natural Killer/Dendritic Cell Axis Defines Responsive Tumor Microenvironments in Melanoma. Nature medicine, 24(8), 1178-1191.

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Identification of Lung ILC2 Cells

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Lung ILC2 cell identification was performed as described previously (47 (link)). Lung tissues were digested in 8 mL RPMI 1640 containing liberase (50 μg/mL) and DNase I (1 μg/mL) for approximately 40 minutes at 37°C. Cell suspensions were filtered through 70 μm cell strainers and washed once with RPMI 1640. For ILC2 cell identification, total lung cell suspensions were blocked with 2.4G2 antibodies and stained with lineage cocktail mAbs: CD3ε (clone 145-2C11) (BioLegend, 100304), CD4 (clone GK1.5) (BioLegend, 100404), CD8α (clone 53-6.7) (Tonbo Biosciences, 30-0081-U500), CD11c (clone N418) (BioLegend, 117304), FceRIα (clone MAR-1) (BioLegend, 134304), NK1.1 (clone PK136) (BioLegend, 108704), CD19 (clone 6D5) (BioLegend, 115504), TER119 (clone TER-119) (BioLegend, 116204), CD5 (clone 53-7.3) (BioLegend, 100604), F4/80 (clone BM8.1) (Tonbo Biosciences, 30-4801-U500), Ly6G (clone RB6-8C5) (Tonbo Biosciences, 30-5931-U500), APC-conjugated streptavidin (BioLegend, 405207), PE-conjugated T1/ST2 (clone DIH9) (BioLegend, 145304), PerCP-Cy5.5-conjugated CD25 (clone PC61) (BioLegend, 102030), V450-conjugated Sca-1 (clone D7) (BD Biosciences, 560653), PE-Cy7-conjugated KLRG1 (clone 2F1/KLRG1) (BioLegend, 138416), APC-Cy7-conjugated CD45, and Fixable Viability Dye eFluor 506.

She L., Barrera G.D., Yan L., Alanazi H.H., Brooks E.G., Dube P.H., Sun Y., Zan H., Chupp D.P., Zhang N., Zhang X., Liu Y, & Li X.D. (2021). STING activation in alveolar macrophages and group 2 innate lymphoid cells suppresses IL-33–driven type 2 immunopathology. JCI Insight, 6(3), e143509.

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Isolation and Characterization of Murine Hematopoietic Stem Cells

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BM was harvested from femurs, tibias, and ilia, and cells were enriched using anti-CD117 magnetic beads (Miltenyi). The c-kit⁺ fraction was stained with antibodies against CD117 (c-kit APC, clone 2B8, Biolegend, dilution 1/100), Sca-1 (APC-Cy7, clone D7, Biolegend, 1/100), CD135 (Flt3 PE-Cy5, clone A2F10, Life technologies, 1/50), CD150 (Slam Pecy7, clone TC15-12F12.2, Biolegend, 1/100), CD48 (Pacific Blue, HM48-1, Biolegend, 1/100), CD16/32 (PercPCy5.5, clone M1/702.4G2, eBioscienceBD Bioscience, 1/100), CD34 (Alexa 700, 1/100), and a lineage cocktail (PE, CD3ε clone 145-2C11; Ly-6G/Ly-6C clone RB6-8C5; CD11b clone M1/70; CD45R/B220 clone RA3-6B2; TER-119, Biolegend, 1/200). The c-kit⁻ fraction was stained with antibodies against CD11b (PercPCy5.5, clone M1/70, eBioscience, 1/100), Ly6C (APC, HK1.4, Thermofisher, 1/200), Ly6G (BV510, RUO, Biolegend, 1/100), Siglec F (PE CF594, RUO, BD 1/100), F4/80 (alexa 700, clone BM8, Ozyme, 1/100), in addition to a lineage cocktail in PeCy7 (B220 clone RA3-6B2 Biolegend, CD3, clone 17A2 Biolegend, CD11c clone N418, eBioscience, NK1.1 clone PK136 Biolegend, Ter119 clone TER-119 BD Biosciences) and DAPI (1/1000) as live/dead marker. All cells were analyzed on a Ze5 (Bio-Rad) plate-reader cytometers. Data analysis was performed using FlowJo v10.2 software (TreeStar) and Prism v9.

Urbanus J., Cosgrove J., Beltman J.B., Elhanati Y., Moral R.A., Conrad C., van Heijst J.W., Tubeuf E., Velds A., Kok L., Merle C., Magnusson J.P., Guyonnet L., Frisén J., Fre S., Walczak A.M., Mora T., Jacobs H., Schumacher T.N, & Perié L. (2023). DRAG in situ barcoding reveals an increased number of HSPCs contributing to myelopoiesis with age. Nature Communications, 14, 2184.

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Multiparametric Immune Cell Analysis

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Spleens and brains were harvested and washed in PBS. A single‐cell suspension was obtained using 70 μm cell strainers. Erythrocytes were lysed in ammonium‐chloride‐potassium lysis buffer (0.15M NH₄Cl, 1 mM KHCO₃, 0.1 mM EDTA, H₂O). Fc receptors were blocked with an anti‐CD16/CD32 antibody (clone 2.4G2; ThermoFisher Scientific) for 15 minutes at 4°C and cells were stained for 30 minutes at 4°C with antibodies against: CD3 (clone 17A2; eBiosciences), NK1.1 (clone PK136; Biolegend), CD49b (clone HMa2; eBiosciences), CD29 (clone HMb1‐1; eBiosciences), CD49d (clone R1‐2; Biolegend) and associated isotype controls. Cells were fixed in PBS‐4% formaldehyde (ThermoFisher Scientific), washed and analysed with a MACSQuant cytometer (Myltenyi Biotec) and with FlowJo software (Treestar).

Petit‐Jentreau L., Glover C, & Coombes J.L. (2018). Parasitized Natural Killer cells do not facilitate the spread of Toxoplasma gondii to the brain. Parasite Immunology, 40(4), e12522.

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Comprehensive Tumor Immunophenotyping by FACS

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FACS analysis was performed using 200-μL tumor or splenocyte single-cell suspensions per antibody panel. Cells were first incubated at 4°C for 30 minutes in PBS with anti-CD16/CD32 Fc block (BD Biosciences) at 1: 100 and live/death staining (Zombie Aqua, BioLegend) at 1: 1,000, or Live/Dead fixable blue dead cell stain (Invitrogen). Next, cells were washed and stained at 4°C for 30 minutes in PBS with antibodies against CD45 (clone 30-F11), B220 (clone RA3–6B2), CD3 (clone 17A2), CD8 (clone 53–6.7), MHC-I (clone AF6–88.5), PD-1 (clone RPM1–30), PD-L1 (clone 10F.9G2), NK1.1 (clone PK136), CD11b (clone M1/70), Ly6G (clone 1A8) and F4/80 (clone BM8), all from BioLegend, at 1 μg/mL. PE-labeled H-2Kb - SIINFEKL pentamer (ProImmune) was used at 2.5 μL per staining. In some sets of experiments, absolute cell numbers were calculated by adding 10 μL counting beads (1 × 10⁶ beads/mL, Spherotech) to each sample before acquisition.

Veerman R.E., Akpinar G.G., Offens A., Steiner L., Larssen P., Lundqvist A., Karlsson M.C, & Gabrielsson S. (2023). Antigen-Loaded Extracellular Vesicles Induce Responsiveness to Anti–PD-1 and Anti–PD-L1 Treatment in a Checkpoint Refractory Melanoma Model. Cancer Immunology Research, 11(2), 217-227.

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Multiparameter Flow Cytometry for Myeloid Cell Analysis

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Single cell suspensions were stained with Fc receptor block (TruStain fcX, Biolegend) along with the appropriate anti-mouse antibodies: CD45 (AF700; clone 30-F11, Biolegend), TNFα (AF700; clone MP6-XT22, Biolegend), CD11b (BV605 or BV650; clone M1/70, Biolegend), Ly6C (BV711; clone HK1.4; Biolegend), Ly6G (APC-Cy7; clone 1A8), CD115 (APC; clone AFS98, Biolegend) or PE-Cy7; AFS98, eBioscience), CD68 (PE; clone FA-11, Biolegend). A lineage gate was used to exclude other cell populations and consisted of the following biotinylated antibodies: CD3 (clone 145-2C11, Biolegend), CD19 (clone 6D5, Biolegend), NK1.1 (clone PK136, Biolegend), Ter119 (clone TER-119, Biolegend), and SiglecF (PE-CF594; E50-2440, BD Biosciences). A fixable stain (LIVE/DEAD Blue, Life Technologies) was used to exclude dead cells. For intracellular staining, cells were first stained for surface markers, fixed in 2% PFA, then permeabilized in 0.5% Saponin (Sigma), and stained for intracellular markers. Fluorescence-minus-one controls were used to validate results. Samples were acquired on a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software v10 (FlowJo LLC, USA).

O'Boyle C., Haley M.J., Lemarchand E., Smith C.J., Allan S.M., Konkel J.E, & Lawrence C.B. (2019). Ligature-induced periodontitis induces systemic inflammation but does not alter acute outcome after stroke in mice. International Journal of Stroke, 15(2), 175-187.

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Multicolor Flow Cytometry of Immune Cells

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LL2 and MC38 cells were purchased from ATCC (Rockville, MD). Cells were cultured in vitro in RPMI-1640 media supplemented with 10% fetal bovine serum, 50 units/mL of penicillin/streptomycin, 2 mM of L-glutamine, 1 mM of sodium pyruvate, and 2 nM of non-essential amino acids, and grown at 37°C. For flow cytometric analyses, antibodies including CD11b (clone M1/70, Biolegend), Gr-1 (Clone RB6–8C5, Biolegend), Ly6C (clone HK1.4, Biolegend), Ly6G (Clone 1A8, Biolegend), RAE-1γ (clone CX1, Biolegend), CD4 (clone GK1.5), CD3ε (clone 145–2C11, BD Bioscience), NK1.1 (clone PK136, Biolegend), Foxp3 (clone PCH101, eBioscience), CD8 (53–6.7, Biolegend), IFNg (XMG1.2, Biolegend) and CD45 (30-F11, Biolegend) were used. Live cell gating was performed using Zombie Aqua Fixable Viability Kit (Biolegend). To detect NKG2D-Fc, we used the PE goat anti-mouse antibodies (Multiple Adsorption, BD Bioscience Cat No: 550589).

Feng P.H., Lam B., Tseng S.H., Kung Y.J., Farmer E., Cheng M.A, & Hung C.F. (2020). NKG2D-Fc fusion protein promotes antitumor immunity through the depletion of immunosuppressive cells. Cancer immunology, immunotherapy : CII, 69(10), 2147-2155.

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Immunohistochemistry and Flow Cytometry Protocol

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Five micron paraffin embedded tissue sections were processed for immuno-histochemistry as previously described (Sur et al., 2012b (link)). Rabbit polyclonal anti-Myc (Santa Cruz, sc-764; RRID:AB_631276) (1:500), Rabbit monoclonal anti Ki-67 (abcam, ab16667; RRID:AB_302459) (1:200), Goat polyclonal anti-Vimentin (Santa Cruz, sc-7557; RRID:AB_793998) (1:500), biotinylated goat anti-Rabbit IgG (Vector Laboratories, BA1000; RRID:AB_2313606) and biotinylated rabbit anti-Goat IgG (Vector Laboratories, BA5000; RRID:AB_2336126) (1:350) antibodies were used. For flow cytometry, single cell suspensions of spleen and bone-marrow and cells from peripheral blood were stained with Fc-block (CD16/CD32 clone 93, Biolegend, 101302, RRID:AB_312801) and subsequently with CD19 (clone 1D3, BD Biosciences, RRID:AB_11154223), TER119 (clone TER119, Biolegend 116210, RRID:AB_313711), CD3ε (clone 145–2 C11, Biolegend 100308, RRID:AB_312673), NK1.1(clone PK136, Biolegend, 108716, RRID:AB_493590), GR1/LY6G (clone RB6-8C5, Biolegend, 108410, RRID:AB_313375), CD4 (clone RM4-5, BD Biosciences, 563747) and CD8a (clone 53–6.7, BD Biosciences, 563332). Dead cells were visualized using Propidium iodide. Samples were analyzed using a BD LSRFortessa instrument.

Dave K., Sur I., Yan J., Zhang J., Kaasinen E., Zhong F., Blaas L., Li X., Kharazi S., Gustafsson C., De Paepe A., Månsson R, & Taipale J. (2017). Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth. eLife, 6, e23382.

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Isolation and Characterization of Liver Immune Cells

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The livers were perfused with phosphate-buffered saline containing 0.2% BSA and dissociated with the gentleMACS™ Dissociator (Miltenyi Biotec, Auburn, CA, USA). The parenchymal cells were removed as pellets after centrifugation at 50×g for 5 min, and the non-parenchymal cells were isolated using 40% and 70% Percoll (GE HealthCare Biosciences, Quebec, Canada). Subsets of liver mononuclear cells were obtained using flow cytometry. Cells were incubated with anti-CD16/32 (clone 93, Biolegend, San Diego, CA, USA) at room temperature for 10 min before staining with fluorochrome-conjugated monoclonal antibodies (mAb) against cell surface markers, including CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8a (clone 53–6.7), NK1.1 (clone PK136), and CD19 (clone 6D5) (Biolegend, San Diego, CA, USA) at 4 °C for 30 min. Stained cells were measured using a flow cytometer (BD FACSVerse) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Wang Y.W., Lin C.I., Chen H.W., Wu J.C, & Chuang Y.H. (2022). Apoptotic biliary epithelial cells and gut dysbiosis in the induction of murine primary biliary cholangitis. Journal of Translational Autoimmunity, 6, 100182.

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Multicolor FACS Analysis of Immune Cells

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Antibodies directed against murine CD3 (clone 145-2C11, Biolegend), CD4 (clone RM4-5, Biolegend), CD8 (clone 53-6.7, Biolegend), NK1.1 (clone PK136, Biolegend), CD62L (clone MEL-14, Biolegend), CD44 (clone IM7, Biolegend), and CD19 (clone 6D5, Biolegend) were used in multicolor FACS analysis. Samples were washed and resuspended in cold flow cytometry staining buffer (1% BSA/PBS); stained for 30 min in the dark before a final wash and acquisition. All samples were acquired on a BD Fortessa Flow cytometer running FACS DIVA software. Analysis was performed using FlowJo X software (TreeStar; OR, United States).

McCormack R.M., de Armas L.R., Shiratsuchi M., Fiorentino D.G., Olsson M.L., Lichtenheld M.G., Morales A., Lyapichev K., Gonzalez L.E., Strbo N., Sukumar N., Stojadinovic O., Plano G.V., Munson G.P., Tomic-Canic M., Kirsner R.S., Russell D.G, & Podack E.R. (2015). Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria. eLife, 4, e06508.

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