Nk1.1 pk136

Manufactured by BioLegend

Sourced in United States

NK1.1 (PK136) is a mouse monoclonal antibody that recognizes the NK1.1 antigen, which is expressed on natural killer (NK) cells and a subset of T cells in mice. It can be used for the identification and isolation of NK cells.

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Lab products found in correlation

Cd11b m1 70, by BioLegend (7 mentions) Ly6g 1a8, by BioLegend (6 mentions) Cd45 30 f11, by BioLegend (5 mentions) Cd11c n418, by BioLegend (5 mentions) Cd19 6d5, by BioLegend (5 mentions) Cd3 17a2, by BioLegend (3 mentions) Cd4 gk1, by BioLegend (3 mentions) Cd11b, by BD (3 mentions) Cd3e 145 2c11, by BioLegend (3 mentions) Foxp3 fjk 16s, by Thermo Fisher Scientific (3 mentions) Cd8 53 6, by BD (3 mentions) Cd11b, by Thermo Fisher Scientific (2 mentions) Cd19 6d5, by Thermo Fisher Scientific (2 mentions) Gr1low, by BD (2 mentions) Gr 1 rb6 8c5, by BioLegend (2 mentions) Facscalibur, by BD (2 mentions) Cd11c, by BD (2 mentions) F4 80, by BD (2 mentions) Ly6c hk1, by BioLegend (2 mentions) Live dead, by Thermo Fisher Scientific (2 mentions)

26 protocols using nk1.1 pk136

Mouse NK Cell Phenotyping and Functional Assays

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All surface antigen staining procedures were performed for 20 min at 4°C in FACS buffer (PBS + 2% FBS). Data were acquired from an LSR Fortessa flow cytometer (BD Biosciences) and analysed using FlowJo v9.9.6 (Tree Star). The antibodies used for surface staining include CD3 (145-2C11), NKp46 (29A1.4) from BD Biosciences, and NK1.1 (PK136) from BioLegend. Antibodies used for in vitro stimulation and killing assays include NKp46 (polyclonal; R&D Systems), NK1.1 (PK136; BioLegend), CD107a (1D4B; BD Biosciences), and IFN-γ (XMG1.2; BD Biosciences). Dead cells were determined using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen). Rat anti-mouse antibodies used for mouse NK cell enrichment obtained from BioLegend include CD3 (17A2), CD19 (6D5), Ter119 (TER-119), Gr-1 (RB6.8C5), CD4 (RM4–5), and CD8 (53–6.7). The secondary antibody used to cross-link mouse NK1.1 antibodies in the calcium flux experiments is goat anti-mouse IgG (H + L) polyclonal antibody (Jackson Immunoresearch).

Luu T.T., Schmied L., Nguyen N.A., Wiel C., Meinke S., Mohammad D.K., Bergö M., Alici E., Kadri N., Ganesan S, & Höglund P. (2021). Short-term IL-15 priming leaves a long-lasting signalling imprint in mouse NK cells independently of a metabolic switch. Life Science Alliance, 4(4), e202000723.

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Multiparameter Flow Cytometry of Tumor Cells

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Tumor tissues were digested both mechanically by chopping with razor blades and chemically with 1mg/mL type IA collagenase (Sigma-Aldrich) for 30 minutes at 37°C. Following digestion, cell suspensions were washed, filtered and stained as previously described (O’Sullivan et al., 2012 (link)). The following antibodies were used: Ly6C (ER-MP20, Serotec), MHCII (M5/114 15.2, eBioscience), Ly6G (1A8, Biolegend), CD8 (53-6.7, eBioscience), CD44 (IM7, Biolegend), CD3 (17A.2, Biolegend), CD4 (GK1.5, Biolegend), CD69 (H1.2F3, Biolegend), Granzyme B (NGZB, eBioscience), IFNγ (XMG 1.2, Biolegend), TCRβ (H57-597, Biolegend), B220 (RA3-6B2, eBioscience), NK1.1 (PK136, Biolegend), CD11b (M1/70, eBioscience), CD45 (30-F11, Biolegend). Stained cell suspensions were analyzed on a BD FACS CANTO II (BD Biosciences).

Saddawi-Konefka R., Seelige R., Gross E.T., Levy E., Searles S.C., Washington A J.r., Santosa E.K., Liu B., O’Sullivan T.E., Harismendy O, & Bui J.D. (2016). Nrf2 induces IL-17D to mediate tumor and virus surveillance. Cell reports, 16(9), 2348-2358.

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Isolation of Lamina Propria Immune Cells

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Colons were dissected, washed with ice-cold PBS and cut into small pieces. Colons pieces were incubated with PBS containing 1 mM DTT, 5 mM EDTA and 10 mM HEPES at 37°C for 30 min with gentle shaking to remove the epithelial layer. The colon segments were further digested in RPMI medium containing 0.5 mg/ml collagenase D at 37°C for 1.5 hr. The supernatant was passed through 70 µm cell strainer and enriched by 37.5% Percoll to isolate lamina propria cells.
The following monoclonal antibodies were used for flow cytometry: CD4 (RM4–5; 14–0042–85), CD11b (M1/70; 48–0112-82) and CD8a (53-6.7; 48–0081–82) from Affymetrix eBioscience, CD19 (6D5; 115512), NK1.1 (PK136; 108708), CD11c (N418; 117306), Gr1 (RB6–8C5; 108426) and F4/80 (BM8; 123109) from BioLegend. The dilution factor for all antibodies was 1:300. The following gating strategies were used: B cells were gated as live cells and CD19⁺. CD4⁺ T cells were gated as live cells, CD4⁺ and CD8⁻. CD8⁺ T cells were gated as live cells, CD8⁺ and CD4⁻. NK cells were gated as live cells and NK1.1⁺. Macrophages were gated as live cells, CD11b⁺, Gr1^low-neg, F4/80⁺ and CD11c⁻. Neutrophils were gated as live cells, CD11b⁺ and Gr1^hi. CD11b⁺CD11c⁺ cells were gated as live cells, CD11b⁺, Gr1^low-neg, CD11c⁺ and F4/80⁻. Flow cytometry data were acquired on a BD FACSCalibur and analysed using TreeStar FlowJo software.

Karki R., Man S.M., Malireddi R.K., Kesavardhana S., Zhu Q., Burton A.R., Sharma B.R., Qi X., Pelletier S., Vogel P., Rosenstiel P, & Kanneganti T.D. (2016). NLRC3 is an inhibitory sensor of PI3K–mTOR pathways in cancer. Nature, 540(7634), 583-587.

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Multi-Omics Immune Cell Profiling

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Cells from tumor or spleen were isolated by grinding through 70-μm filters and stained with the following fluorochrome-conjugated antibodies: CD45 (30-F11, BioLegend, 103116); 7-AAD (Tonbo Biosciences, 13-6993-T200); CD3 T cells: CD3e (145-2C11, BioLegend, 100305); CD8 T cells: CD8a (53-6.7, BioLegend, 100708); CD4 T cells (GK1.5, BioLegend,100412); neutrophils and monocytes: Ly6C (HK1.4, BioLegend, 115506) and CD11b (M1/70, BioLegend, 101216); natural killer cells: NK1.1 (PK136, BioLegend, 108722), B cells: CD19 (6D5, BioLegend, 115506). For the intracellular IFNγ staining, cells were incubated for 5 h at 37°C in complete RPMI 1640 containing 2 µM Monensin, 50 ng/mL PMA, 1 µg/mL Ionomycin, followed by incubation in BD Perm buffer for 30 minutes at 4°C, washed by BD wash buffer and stained with the antibody IFNγ (XMG1.2, Biolegend, 505806). The stained cells were acquired on the Invitrogen™ Attune™ NxT acoustic flow cytometer system and the data were analyzed with using FlowJo software (BD Bioscience).

Ye S., Zhu Y., Zhong D., Song X., Li J., Xiao F., Huang Z., Zhang W., Wu M., Zhang K., Xiang F.L, & Xu J. (2023). G protein-coupled receptor GPR68 inhibits lymphocyte infiltration and contributes to gender-dependent melanoma growth. Frontiers in Oncology, 13, 1202750.

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Profiling Lung Immune Cell Subsets

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The collected lung immune cells were incubated with anti-CD16/CD32 (2.4G2) and in the dark for 30 min at 4°C with various combinations of fluorescent-conjugated antibodies against CD11c (N418), CD11b (M1/70), F4/80 (BM8), Ly6C (HK1.4), Ly6G (1A8), PDCA1 (HM1.2), CD45 (30-F11), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD19 (6D5), and NK1.1 (PK136) (all from BioLegend). DAPI or 7-ADD were used to separate live and dead cells. The cells were sorted using a FACS Verse flow cytometer (BD Bioscience), and the data were analyzed with FlowJo version 10.5.3 software (BD Bioscience) (Supplementary Figure 1).

Seo S.U., Jeong J.H., Baek B.S., Choi J.M., Choi Y.S., Ko H.J, & Kweon M.N. (2021). Bleomycin-Induced Lung Injury Increases Resistance to Influenza Virus Infection in a Type I Interferon-Dependent Manner. Frontiers in Immunology, 12, 697162.

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Multiparametric Flow Cytometry Immunophenotyping

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Single-cell suspensions were stained in an FACS buffer (0.1% BSA and 2 mM EDTA in PBS (Sigma Aldrich)) using optimised concentrations of Fc Block (2.4G2; BD Biosciences) and then combinations of the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs) for peripheral blood samples: IA-IE (M5/114-15.2) from BD Biosciences, Ly6C (HK1.4) from eBioscience and F4/80 (BM8) from BioLegend (NSW, Australia); for spleen samples: CD11c (N418), CD4 (GK1.5), B220 (RA3-6B2), CD8a (53-6.7), F4/80 (BM8), IA-IE (M5/114-15.2), Ly6C (HK1.4). To assist in identifying rare myeloid populations, cells were also stained with a lineage (Lin) mixture containing biotinylated mAbs: CD3e (145-2C11), CD19 (1D3) from BD Biosciences, and Ly6G (1A8) and NK1.1 (PK136) from BioLegend. Lin mAbs were detected using streptavidin–Brilliant Violet 421 (BD Biosciences) (Supplementary Materials Figure S1). Data were collected on the ACCURI C6 flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, version 9).

Shi H., Lo T.H., Ma D., Condor B., Lesmana B., Parungao R.J., Tsai K.H., Kim S., Chen H.T., Silveira P.A., Li Z., Cooper M.S., Simanainen U., Handelsman D.J., Maitz P.K, & Wang Y. (2020). Dihydrotestosterone (DHT) Enhances Wound Healing of Major Burn Injury by Accelerating Resolution of Inflammation in Mice. International Journal of Molecular Sciences, 21(17), 6231.

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Multicolor Flow Cytometry for Lung Immune Cells

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The lung cells were isolated by digestion with 400 U/ml collagenase D in PBS containing Ca²⁺ Mg²⁺ at 37°C for 25 min, followed by filtration through a 70-µm cell strainer. The lung cells or BAL cells were resuspended in PBS containing 2% FBS. Nonspecific binding was blocked by incubating with 12.5 µg/ml Fc Block (93, eBioscience) for 15 min at 4°C. Samples were incubated with anti-mouse antibodies including FITC-conjugated CD11b (M1/70, BioLegend), CD3 (17A2, BioLegend), PE-conjugated CD64 (X54-5/7.1, BioLegend), NK1.1 (PK136, BioLegend), PerCP-conjugated CD45 (30-F11, BioLegend), PE/Cy-7-conjugated CD11c (N418, BioLegend), APC-conjugated MHCII (M5/114.15.2, BioLegend), Ly6G (1A8, BioLegend), APC/Cy-7-conjugated CD19 (6D5, BioLegend), CD11b (M1/70, BD Pharmingen), and Pacific Blue-conjugated Siglec-F (E50-2440, BD Pharmingen) for 30 min at 4°C. Samples were washed twice with FACS buffer, and flow cytometry was performed after gating on the CD45⁺ leukocyte population using a BD Canto II flow cytometer (BD biosciences). Results were analyzed with FlowJo software ver. 9.3.2.

Lee M.R., Shim D., Yoon J., Jang H.S., Oh S.W., Suh S.H., Choi J.H, & Oh G.T. (2014). Retnla Overexpression Attenuates Allergic Inflammation of the Airway. PLoS ONE, 9(11), e112666.

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Multiparametric Flow Cytometry Profiling

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Cells were isolated from the indicated tissues. Single-cell suspensions were stained with CD16/32 and with indicated fluorochrome-conjugated antibodies. Live/Dead Fixable Violet Cell Stain Kit (Invitrogen) was used to exclude non-viable cells. Multi-laser, flow cytometry analysis procedures were done at the University of Pennsylvania Flow Cytometry and Cell Sorting Facility using BD LSRII cell analyzers running FACSDiva software (BD Biosciences). Two-laser, flow cytometry analyses were done at the University of Pennsylvania iPS Cell Core using BD Accuri C6 instruments. FlowJo software (v.10 TreeStar) was used for data analysis and graphics rendering. All fluorochrome-conjugated antibodies used are listed as follows (Clone, Company, Catalog Number): CD11b (M1/70, Biolegend, 101255); CD11c (N418, Biolegend, 117318); CD16/32 (93, Biolegend, 101319); CD16/32 (93, eBiosciences, 56D0161D80); CD19 (6D5, Biolegend, 115510); CD3ε (145D2C11, Biolegend, 100304); CD4 (GK1.5, Biolegend, 100406); CD45 (30-F11, Biolegend, 103121 or 103151), CD8a (53D6.7, Biolegend, 100725); Foxp3 (FJK-16s, eBiosciences, 50-5773-82); Ly-6G (1A8, Biolegend, 127624); Live/Dead (N/A, Thermofisher, LD34966); NK1.1 (PK136, Biolegend, 108745); RORγt (B2D, eBiosciences, 12-6981-82); Siglec-F (E50D2440, BD, 562757); TCRγδ (UC7-13D5, Biolegend, 107504)

Tang A.T., Choi J.P., Kotzin J.J., Yang Y., Hong C.C., Hobson N., Girard R., Zeineddine H.A., Lightle R., Moore T., Cao Y., Shenkar R., Chen M., Mericko P., Yang J., Li L., Tanes C., Kobuley D., Võsa U., Whitehead K.J., Li D.Y., Franke L., Hart B., Schwaninger M., Henao-Mejia J., Morrison L., Kim H., Awad I.A., Zheng X, & Kahn M.L. (2017). Endothelial TLR4 and the microbiome drive cerebral cavernous malformations. Nature, 545(7654), 305-310.

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Phenotyping Immune Cells in Infection

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Peritoneal exudate cells (PEC), peripheral blood, and spleen (SP) obtained from infected and PBS-injected mice were stained with anti-Ly6B.2 (7/4) (abcam), anti-Ly6G (1A8), -CD11b (M1/70), -CD49d (R1-2), -CD29 (HMβ1-1), -CD182 (CXCR2; SA044G4), -CD184 (CXCR4; L276F12), -CD121 (IL-1R; JAMA147), -CD45 (30-F11), -TCRβ (H57-597), -CD4 (GK1.5), -CD8 (53-6.7), -Ly6C (HK1.4), -B220 (RA3-6B2), and NK1.1 (PK136) (all from Biolegend). For intracellular cytokine staining (ICS), erythrocyte-depleted splenocytes were resuspended in RPMI 1640 with 10% FCS and 1 μg/ml Golgiplug (BD), monensin (eBioscience), and anti-CD107a (1D4B) (BD). After re-stimulation with HIV-1 peptides (6 h, 37°C, 5% CO₂), splenocytes were stained for surface markers with anti-CD3 (145-2C11), -CD4 (GK1.5), and -CD8 (53-6.7) (all from BD), fixed, permeabilized (Cytofix/Cytoperm kit; BD), and stained intracellularly with anti-IFN-γ (XMG 1.2) and -TNF (MP6-X722) (all from BD). Cells were acquired using a GALLIOS (Beckman Coulter) or LSRII (BD) flow cytometer and data were analyzed with FlowJo software v.9.9.5 and 10.5.3.

Di Pilato M., Palomino-Segura M., Mejías-Pérez E., Gómez C.E., Rubio-Ponce A., D’Antuono R., Pizzagalli D.U., Pérez P., Kfuri-Rubens R., Benguría A., Dopazo A., Ballesteros I., Sorzano C.O., Hidalgo A., Esteban M, & Gonzalez S.F. (2021). Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection. NPJ Vaccines, 6, 52.

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Monitoring NK Cells in MCMV Infection

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Fc receptors were blocked with anti-CD16+CD32 mAb (2.4G2) before staining. To monitor NK cells in peripheral blood weekly after MCMV infection, 50 μL of heparinized blood was lysed by ACK buffer and stained with anti-TCRβ (H57-597; BioLegend), -NK1.1 (PK136; BioLegend), -Ly49H (3D10; eBioscience), -CD45.1 (A20; BioLegend), -CD45.2 (104; Tonbo Biosciences) and -KLRG1 (2F1; BioLegend). Cells were analyzed on a BD LSR II flow cytometer using FlowJo software (Tree Star).

Kamimura Y, & Lanier L.L. (2015). Homeostatic control of memory cell progenitors in the NK cell lineage. Cell reports, 10(2), 280-291.

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