Anti nk1

Manufactured by BD

Sourced in United States

Anti-NK1.1 is a monoclonal antibody used for the detection and analysis of natural killer (NK) cells in laboratory research. It binds specifically to the NK1.1 antigen, a cell surface marker expressed on NK cells in certain mouse strains.

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Lab products found in correlation

Anti b220, by BD (7 mentions) Anti cd8, by BD (7 mentions) Anti gr 1, by BD (6 mentions) Anti cd11b, by BD (6 mentions) Anti cd4, by BD (5 mentions) Anti ter119, by BD (5 mentions) Anti cd3, by BD (4 mentions) Anti cd69, by BD (3 mentions) Anti cd45, by BioLegend (3 mentions) Anti cd11c, by BD (3 mentions) Anti cd19, by BD (3 mentions) Anti cd3e, by BD (3 mentions) Facscalibur, by BD (2 mentions) Anti cd25, by BD (2 mentions) Anti cd44, by BD (2 mentions) Anti cd19, by Cytek Biosciences (2 mentions) Anti cd3, by BioLegend (2 mentions) Anti ter119, by BioLegend (2 mentions) Anti mac 1, by BD (2 mentions) Apc conjugated anti il 7rα, by Miltenyi Biotec (2 mentions)

17 protocols using anti nk1

Isolation and Flow Cytometric Analysis of Lymphocytes

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Spleen, bone marrow and thymus were isolated from age-matched 26 week-old WT and Rag-2^−/− mice for flow cytometric analysis. The organs were homogenized by passing through 40 µm cell strainer (SPL Life Science). After removing RBC from the isolated cells using a RBC lysis buffer (Invitrogen, USA) and cell counting, 1×10⁷ cells were stained with the following antibodies (all antibodies except for anti-NK1.1 (eBioscience, USA) were from BD Bioscience, USA and 1:200 used for all antibodies); V450-anti-CD3ε (500A2) and either FITC-anti-CD4 (RM4-5) or BV605-anti-CD8a (53-6.7) for T cells, V450-anti-IgM (R6-60.2) and PerCP-anti-B220 (RA3-6B2) for B cells, and anti-CD3ε and APC-anti-NK1.1 (PK136;) for NK cells. 20,000 cells were analyzed using a FACSCalibur (BD biosciences). In flow cytometry, cells were selected based on forward and side scatter properties after gating for singlets and each lymphocyte population was gated using fluorescence intensity of the aforementioned antibodies against cell surface markers. Unstained cells were used to set appropriate negative gates by determining the background fluorescence levels, and single stained cells were used as a control to remove spectral overlap between different fluorophores.

Kim J.I., Park J.S., Kim H., Ryu S.K., Kwak J., Kwon E., Yun J.W., Nam K.T., Lee H.W, & Kang B.C. (2018). CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice. Laboratory Animal Research, 34(4), 166-175.

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Spleen Cell Isolation and Characterization

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The 0.2 g spleens were washed with PBS solution and passed through a cell strainer, then the red blood cells were removed from the suspension with red blood cell lysis buffer. Suspensions were then centrifuged, and supernatants were removed. Next, cell sedimentations were suspended with PBS again, and the concentration of cells is about 10⁶/mL. The cell suspensions were stained with antibodies (anti- CD3⁺, anti-CD3⁺CD4⁺, anti-CD3⁺CD8⁺, anti-CD19, and anti-NK1.1, purchased from BD Biosciences, Shanghai, China). The cells (CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, B, and NKT cells) were analyzed by flow cytometry (BD FACSVerse, BD Biosciences, Shanghai, China).

Zhou X., Zhao L., Luo J., Tang H., Xu M., Wang Y., Yang X., Chen H., Li Y., Ye G., Shi F., Lv C, & Jing B. (2019). The Toxic Effects and Mechanisms of Nano-Cu on the Spleen of Rats. International Journal of Molecular Sciences, 20(6), 1469.

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Multiparameter Analysis of Hepatic Immune Cells

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Isolated liver MNCs were resuspended in PBS containing 1% BSA. NKT cells and T cells were determined by staining with anti-CD3, anti-CD4, anti-NK1.1, and anti-CD69 (BD Pharmingen). For detection of IFN-γ and LC3, cells with surface staining were fixed and permeabilized by Cytofix/Cytoperm kit (eBioscience), further stained with anti-IFN-γ or anti-LC3 antibodies. To evaluate nuclear factor-κB (NF-κB) activity in T cells, hepatic MNCs from Con A-injected mice were permeabilized by methanol followed stained with fluorescence-conjugated antibodies against phospho-NF-κB p65 and CD3. T cell apoptosis was monitored by FACS analysis using Annexin V/propidium iodide (PI) staining, according to the manufacturer’s instruction (Keygen Biotech, Nanjing, China).

Li Y., Tang Y., Wang S., Zhou J., Zhou J., Lu X., Bai X., Wang X.Y., Chen Z, & Zuo D. (2016). Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation. Frontiers in Immunology, 7, 350.

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Multicolor Flow Cytometry Protocol

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Cell membrane staining was performed with fluorochrome-conjugated mAbs, after blocking with anti-FcγR (clone 2.4G2) mAb. The following mAbs were used (clone indicated in parentheses): anti-CD11c (HL3), anti-I-Ab or MHCII (M5/114.15.2) anti-CD40 (HM40-3), anti-CD11b (M1/70), anti-CD8α (53-6.7), anti-CD3 (145-2C11), anti-NK1.1 (PK136), anti-c-kit (2B8), anti-TCRβ (H57-597); anti-CD4 (RM4-4); anti-CD8β.2 (53-5.8); anti-CXCR4 (2B11) [from BD Biosciences; Biolegend, San Diego, CA, USA; Miltenyi Biotec; eBioscience; conjugated with FITC, phycoerythrin (PE), peridinin chlorophyll protein (PerCP)-Cy5.5, PE-Cy7, Alexa 647, APC, APC-Vio770; APC-H7]. Dead cells were excluded with Propidium Iodide (PI, Sigma-Aldrich). Samples were analyzed by FACSCanto I and II (BD Biosciences). Data were analyzed using FlowJo software, v.9.7.6 (FlowJo, Ashland, OR, USA).

Barroeta Seijas A.B., Simonetti S., Vitale S., Runci D., Quinci A.C., Soriani A., Criscuoli M., Filippi I., Naldini A., Sacchetti F.M., Tarantino U., Oliva F., Piccirilli E., Santoni A, & Di Rosa F. (2017). GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells. Frontiers in Immunology, 8, 147.

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Multiparameter Flow Cytometry Analysis

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Single cell suspensions were prepared and washed in ISCOVES medium. After centrifugation, erythrocytes were lysed in Red Cell Removal Buffer (RCRB; 156 mM NH₄Cl, 10 μM EDTA, 1 mM Na₂CO₃) and FCS was subsequently added (3 (link)). Cells were counted and 10⁶ cells per sample were used for staining. Cells were washed twice in PBS containing 3% FCS and 0.1% NaN₃ and were subsequently stained with optimal concentrations of anti-IgM, anti-IgD, anti-B220, anti-CD25, anti-CD21, anti-CD43, anti-Thy 1.2, anti-NK1.1, or anti-CD138 (all from BD Bioscience). Fluorochrome-labeled streptavidin and the apoptosis marker merocyanine (Sigma Aldrich, Munich, Germany) were incubated separately. Samples were subsequently acquired on a FACSCalibur (BD Bioscience) and analyzed using the CellQuest software (BD Bioscience).

Müller U., Schaub G.A., Mossmann H., Köhler G., Carsetti R, & Hölscher C. (2018). Immunosuppression in Experimental Chagas Disease Is Mediated by an Alteration of Bone Marrow Stromal Cell Function During the Acute Phase of Infection. Frontiers in Immunology, 9, 2794.

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Comprehensive Immune Cell Profiling by Flow Cytometry

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Cell surface staining were performed with the following antibodies: anti-CD3ε, and anti-CD4, anti-CD11b, anti-CD11c, anti-CD19, anti-B220, and anti-Ly-6G (TONBO Bioscience, San Diego, CA, USA); anti-IL-33R (ST2), anti-TCRγδ, anti-CD90.2, and anti-CD45 (BioLegend, San Diego, CA, USA); anti-TCRβ, anti-CD25, anti-CD44, anti-CD62L, anti-CD69, anti-CD103, anti-NK1.1, and anti-TER119 (BD Bioscience); and anti-CD127 (eBioscience, San Diego, CA, USA). The same procedures were conducted with the following antibodies for the intracellular staining: anti-IL-4 and anti-IL-5 (BD Bioscience); and anti-IL-13 (eBioscience). Fixable Viability Dye (eBioscience) was used to exclude dead cells. All samples were analyzed using the FACS Verse (BD Bioscience) and Flow Jo software programs (Tree Star Inc., San Carlos, CA, USA).

Mato N., Hirahara K., Ichikawa T., Kumagai J., Nakayama M., Yamasawa H., Bando M., Hagiwara K., Sugiyama Y, & Nakayama T. (2017). Memory-type ST2+CD4+ T cells participate in the steroid-resistant pathology of eosinophilic pneumonia. Scientific Reports, 7, 6805.

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Murine Bone Marrow Immunophenotyping

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Femora and tibiae were isolated from 6 month old mice and flushed with Hanks’ Balanced Salt Solution containing 1% FBS. The marrow cell suspension was then stained and analyzed by flow cytometry. The following antibodies were used: anti-CD43-PE (BD Pharmingen, San Jose, CA), anti-c-Kit-APCCy7 (BD Pharmingen), anti-Sca-1-FITC (BD Pharmingen), anti-B220-FITC (BD Pharmingen), anti-IL-7rα PECy7 (eBioscience, San Diego, CA, USA), anti-CD19-APCCy7 (BD Pharmingen), anti-IgM-APC (BD Pharmingen), anti-CD34-APC (BD Pharmingen). To analyze Lin⁻ cells, total bone marrow cells were stained with a biotinylated Lin⁺ antibody cocktail of anti-CD4, anti-CD8, anti-NK1.1, anti-CD3, anti-CD11c, anti-B220, anti-CD19, anti-Gr1, anti-CD11b and anti-Ter119 (BD Pharmingen), followed by streptavidin-Pacific Blue (BD Pharmingen). FACS analyses were performed on a FACS Canto II running with the FACSDiva software (Becton Dickinson, Franklin Lakes, NJ, USA). In all analyses, propidium iodide was used to exclude dead cells. FACS analysis was done using the FlowJo (Treestar Inc., Ashland, OR, USA) software.

Remoli C., Michienzi S., Sacchetti B., Di Consiglio A., Cersosimo S., Spica E., Robey P.G., Holmbeck K., Cumano A., Boyde A., Davis G., Saggio I., Riminucci M, & Bianco P. (2015). Osteoblast-specific expression of the Fibrous Dysplasia (FD) causing mutation, GsαR201C produces a high bone mass phenotype but does not reproduce FD in the mouse. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 30(6), 1030-1043.

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Immunophenotyping of Hematopoietic Cells

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Flow cytometric analysis of the OP9 cells and primary BM cells were performed with the use of FACSCanto (Becton Dickinson, San Jose, CA). The following antibodies were used: anti-PDGFRβ, anti-CD31, anti-CD45, anti-Ter119, anti-CD54 (Biolegend), anti-CD3, anti-CD4, anti-CD8, anti-CD34, anti-Mac-1, anti-Gr-1, anti-B220, and anti-TER-119, anti-NK.1.1, anti-CD106 anti-hCDRA (BD Biosciences), and anti-CD61 anti-CD140a (eBioscience) anti-CD44 anti-hCD45RO (Pharmingen), anti-Notch-1, anti-Notch-4, anti-Jagged-1 (eBioscience), anti-Notch-2, anti-Notch-3, anti-Dll-1, and anti-jagged-2 (Biolegend).

Supper E., Tahir S., Imai T., Inoue J, & Minato N. (2015). Modification of Gene Expression, Proliferation, and Function of OP9 Stroma Cells by Bcr-Abl-Expressing Leukemia Cells. PLoS ONE, 10(7), e0134026.

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Lung Innate Lymphoid Cell Type 2 Isolation

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The cells isolated from digested lungs were stained with biotin-conjugated antibody mixtures for lineage markers (CD4, CD5, CD8, CD11c, CD11b, CD19, NK1.1, Gr-1, TER119, FcεRI, and B220), Pacific blue-conjugated anti-Sca-1, PECy7-conjugated c-Kit (CD117), APC-conjugated anti-IL-7Rα (CD127), FITC conjugated anti-T1/ST2, APC-Cy7-conjugated anti-CD25, and PE conjugated anti-streptavidin, and analyzed using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin⁻Sca⁺c-Kit⁺IL-7R⁺CD25⁺ST2^dim cells were identified as lung ILC2s [21 (link), 22 (link)]. The data were analyzed using FlowJo (TreeStar, Ashland, OR, USA). APC-Cy7-conjugated anti-CD25, Pacific blue-conjugated anti-Sca-1, biotin-conjugated anti-CD4, anti-CD5, anti-CD8, anti-CD11b, anti-NK1.1, anti-Gr-1, anti-TER119, and anti-B220, and PE-conjugated anti-streptavidin antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-T1/ST2 was from MD Bioscience (St Paul, MN, USA). APC-conjugated anti-IL-7Rα and biotin-conjugated anti-FcεRI antibodies were from BioLegend (San Diego, CA, USA). PECy7-conjugated c-Kit was from eBioscience (La Jolla, CA, USA). Biotin-conjugated anti-CD11c and anti-CD19 were from TONBO Biosciences (San Diego, CA, USA).

Morichika D., Taniguchi A., Oda N., Fujii U., Senoo S., Itano J., Kanehiro A., Kitaguchi Y., Yasuo M., Hanaoka M., Satoh T., Akira S., Kiura K., Maeda Y, & Miyahara N. (2021). Loss of IL-33 enhances elastase-induced and cigarette smoke extract-induced emphysema in mice. Respiratory Research, 22, 150.

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Immune Profiling of Tumor Microenvironment

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Myeloid and lymphoid subsets were isolated from the tumors and quantitatively analyzed as previously described (Chowdhury et al., 2019 (link)) with some modifications. Briefly, after mechanical homogenization, the tumors were digested with collagenase A (1 mg ml−1; Roche) and DNase I (0.5 μg ml−1; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% L-glutamine, 1% penicillin–streptomycin and 10 mM HEPES) for 45 minutes shaking (150 rpm) at 37 °C. Cells were filtered through 100 μm cell strainers, washed in isolation buffer and stained. Myeloid cells were stained immediately, and lymphoid subset underwent a separation gradient using Percoll (67%, 40%), followed by staining. Dead cells were excluded by staining with Ghost Dye cell viability reagent. Extracellular antibodies included: anti-B220 (BD) (1:200), anti-CD19 (Tonbo) (1:200), anti-CD45 (BD and Biolegend) (1:400), anti-CD4 (BD) (1:400), anti-CD8 (Tonbo) (1:400), anti-NK1.1 (BD) (1:300), anti-CD11b (BD) (1:500), anti-CD11c (BD) (1:200), anti-F4/80 (Tonbo) (1:500), and anti-MHC class II (Tonbo) (1:400) antibodies. Intracellular antibodies included: anti-CD3e (BD) (1:400), anti-TCRβ (BD) (1:300) and anti-FOXP3 (Thermo) (1:300). Cells were fixed using the FOXP3/transcription factor staining buffer set (Tonbo) according to the manufacturer’s protocol. Samples were analyzed using a BD LSRFortessa cell analyzer.

Affo S., Nair A., Brundu F., Ravichandra A., Bhattacharjee S., Matsuda M., Chin L., Filliol A., Wen W., Song X., Decker A., Worley J., Caviglia J.M., Yu L., Yin D., Saito Y., Savage T., Wells R.G., Mack M., Zender L., Arpaia N., Remotti H.E., Rabadan R., Sims P., Leblond A.L., Weber A., Riener M.O., Stockwell B.R., Gaublomme J., Llovet J.M., Kalluri R., Michalopoulos G.K., Seki E., Sia D., Chen X., Califano A, & Schwabe R.F. (2021). Promotion of Cholangiocarcinoma Growth by Diverse Cancer-associated Fibroblast Subpopulations. Cancer cell, 39(6), 866-882.e11.

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