Nk1.1 pe cy7

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NK1.1-PE-Cy7 is a fluorochrome-conjugated monoclonal antibody that binds to the NK1.1 antigen expressed on natural killer cells in mice. It is suitable for flow cytometric analysis.

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NK1.1-PE (17 citations) Anti-NK1.1- PE-Cy7 (3 citations) NK1.1-PE-Cy7 (clone PKI 36, (2 citations)

16 protocols using nk1.1 pe cy7

Characterizing T Cell Subsets in Mice

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Single cell suspensions were prepared from the thymus, spleen and lymph nodes of busulfan chimeric mice, wildtype control mice, or germ free mice. Cells were stained with the following monoclonal antibodies and cell dyes: CD45.1 FITC, CD45.2 FITC, CD45.2 AlexaFluor700, TCR-

β

APC, CD4⁺ PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE, CD25 eFluor450, CD25 PE-Cy7, CD62L eFluor450, NK1.1 PE-Cy7 (all eBioscience), CD45.1 BV650, CD45.2 PE-Dazzle, TCR-

β

PerCP-Cy5.5 CD4⁺ BV711, CD44 BV785, CD25 BV650 (all Biolegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD nearIR and LIVE/DEAD blue viability dyes. For Ki67 staining, cells were fixed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set and stained with either anti-mouse Ki67 FITC or PE (both eBioscience). Cells were acquired on a BD LSR-II or a BD LSR-Fortessa flow cytometer and analysed with Flowjo software (Treestar). Conventional CD4⁺ cells were identified as live TCR-

β

+ CD4⁺ CD25- NK1.1-, and then CD44 and CD62L were used to identify EM (CD44+CD62L-) and CM (CD44+CD62L+) subsets.

Hogan T., Nowicka M., Cownden D., Pearson C.F., Yates A.J, & Seddon B. (2019). Differential impact of self and environmental antigens on the ontogeny and maintenance of CD4+ T cell memory. eLife, 8, e48901.

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Isolation and Phenotyping of Immune Cells

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Mononuclear cells from liver and spleen and stromal vascular cells (SVC) from AT (epididymal and perirenal depots) were isolated as described previously (Stefanovic‐Racic et al. 2012). Cell suspensions (2 × 10⁶ cells/sample) were preincubated with anti‐CD16/32 (Fc “blocking” antibodies [Abs]) for 15 min at 4°C, then stained with either fluorescent‐labeled Abs or IgG isotype controls for 30 min at 4°C. The following Abs were used: CD8/PerCP and CD45/PerCP (BD Biosciences, San Jose, CA), CD4/FITC, CD62L/PE, NK1.1/PeCy7, B220/v450, CD3/APC, CD86/FITC, MHC2/PE, CD11b/PeCy7, B220/v450, CD11c/APC, and F4/80/Alexa780 (eBiosciences, San Diego, CA). Following incubation with Abs, liver and AT cells were incubated in Aquaporin dye (Invitrogen, Grand Island, NY) for 15 min, to distinguish live from dead cells, then fixed in 4% paraformaldehyde (Fisher, Waltham, MA) before being analyzed using a FACSCalibur flow cytometer and FACSDiva software (BD Biosciences). A proportion of up to 1% false‐positive events were accepted in the isotype control samples.

Krishna K.B., Stefanovic‐Racic M., Dedousis N., Sipula I, & O'Doherty R.M. (2016). Similar degrees of obesity induced by diet or aging cause strikingly different immunologic and metabolic outcomes. Physiological Reports, 4(6), e12708.

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Flow Cytometric Analysis of Aortic and Blood Cells

Cited 1 time

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Flow cytometry of aortas and whole blood was performed as described previously [33 (link), 68 (link)]. Briefly, cleaned aortic vessels were digested with liberase™ (1 mg/mL, Roche, Basel, Switzerland) for 30 min at 37 °C and passed through a cell strainer (70 μm) to yield a single-cell suspension. Single-cell suspensions were treated with Fc-block (anti-CD16/CD32), washed, and surface stained with CD45 APC-efluor 780 (#47-0451-82, 2 µg/mL), NK1.1 PE-Cy7 (#25-5941-81, 2 µg/mL), F4/80 APC (#17-4801-82, 4 µg/mL) from eBioscience (San Diego, CA) and TCR-β V450 (#560706, 2 µg/mL), CD11b PE (#553311, 2 µg/mL), Ly6G FITC (#551460, 5 µg/mL), and Ly6C PerCP-Cy.5.5 (#560525, 2 µg/mL) from BD Biosciences (San Diego, CA). Dead cells were excluded by staining with Fixable Viability Dye eFluor506 (#65–0866-14, 1:1000, eBioscience, San Diego, CA). Based on a live gate, events were acquired and analyzed using a BD FACS CANTO II flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and FACSDiva software (Becton Dickinson, Franklin Lakes, NJ), respectively. Gating was performed according to a previously published strategy [63 (link)].

Frenis K., Helmstädter J., Ruan Y., Schramm E., Kalinovic S., Kröller-Schön S., Bayo Jimenez M.T., Hahad O., Oelze M., Jiang S., Wenzel P., Sommer C.J., Frauenknecht K.B., Waisman A., Gericke A., Daiber A., Münzel T, & Steven S. (2021). Ablation of lysozyme M-positive cells prevents aircraft noise-induced vascular damage without improving cerebral side effects. Basic Research in Cardiology, 116(1), 31.

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Immune Cell Phenotyping by Flow Cytometry

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The isolated immune cells were resuspended in 0.1 mM PBS and passed through a 70-μm strainer (BD Biosciences, NJ,USA). Samples were analyzed with the following antibodies: anti-human CD14 FITC (325604, Biolegend, San Diego, CA, USA); anti-human Tim-3 PE (345006, Biolegend, San Diego, CA, USA); anti-mouse CD45 APC-eFlour780 (47-0451-82, eBioscience, San Diego, CA, USA), anti-mouse F4/80 PE-eFlour610 (61-4801-82, eBioscience, San Diego, CA, USA); anti-mouse CD11b PE-Cy7 (25-0112-82, eBioscience, San Diego, CA, USA), CD3 APC (100236, Biolegend, San Diego, CA, USA), CD4 percycy5.5 (103132, Biolegend, San Diego, CA, USA), CD8 FITC (100706, Biolegend, San Diego, CA, USA), CD11c APC (17-0114-81, eBioscience, San Diego, CA, USA), NK1.1 pecy7 (25-5941-82, eBioscience, San Diego, CA, USA), anti-mouse Tim-3 PE (12-5870-82, eBioscience, San Diego, CA, USA). Antibodies and their isotype-matched negative control antibodies were incubated with cells at 4 °C for 30 min in dark. Cells were washed with 0.1 mM PBS. The samples were subjected and detected by a Beckman CytoFLEX FCM, and the data were analyzed by CytExpert 2.0 software.

Yang H., Xie T., Li D., Du X., Wang T., Li C., Song X., Xu L., Yi F., Liang X., Gao L., Yang X, & Ma C. (2019). Tim-3 aggravates podocyte injury in diabetic nephropathy by promoting macrophage activation via the NF-κB/TNF-α pathway. Molecular Metabolism, 23, 24-36.

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Phenotypic analysis of lymphocyte subsets

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Single-cell suspensions were prepared from the thymus, spleen and lymph nodes of busulfan chimeric mice, wildtype control mice, or germ-free mice. Cells were stained with the following monoclonal antibodies and cell dyes: CD45.1 FITC, CD45.2 FITC, CD45.2 AlexaFluor700, TCR-

β

APC, CD4⁺ PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE, CD25 eFluor450, CD25 PE-Cy7, CD62L eFluor450, NK1.1 PE-Cy7 (all eBioscience), CD45.1 BV650, CD45.2 PE-Dazzle, TCR-

β

PerCP-Cy5.5 CD4⁺ BV711, CD44 BV785, CD25 BV650 (all Biolegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD near-IR and LIVE/DEAD blue viability dyes. For Ki67 staining, cells were fixed using the eBioscience Foxp3 /Transcription Factor Staining Buffer Set and stained with either anti-mouse Ki67 FITC or PE (both eBioscience). Cells were acquired on a BD LSR-Fortessa flow cytometer and analysed with Flowjo software (Treestar). See Figure 1—figure supplement 1 for the gating strategy used to identify mature single positive thymocytes and peripheral naive subsets, and gates to measure Ki67 frequencies.

Rane S., Hogan T., Lee E., Seddon B, & Yates A.J. (2022). Towards a unified model of naive T cell dynamics across the lifespan. eLife, 11, e78168.

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Immunophenotyping of Liver Infiltrates

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Purified liver immune infiltrates were blocked for non-specific staining in 2% BSA and with anti-CD16 (5 μg/mL) for 20′ at 4°C. Afterwards, cells were stained in 2% BSA with CD4-Violet421 (1:100, Pharminogen), CD8-PE-Cy7 (1:100, Biolegend), F4/80- Fitc (1:100, eBiosciences), CD3-e450 (eBiosciences), NK1.1- PE-Cy7 (eBiosciences), CD19-Violet421 (1:100, Biolegend) and B220-PerCP (1:100, Biolegend), CD11c-Violet 421 (1:100, Biolegend), MHCII-PerCP (1:100, Biolegend). At last, the cells were washed twice in 2% BSA and analyzed by flow cytometry using Attune (Invitrogen), and FlowJo.

Dibra D., Xia X., Mitra A., Cutrera J.J., Lozano G, & Li S. (2016). Mutant p53 in concert with an IL27 Receptor α deficiency causes spontaneous liver inflammation, fibrosis, and steatosis. Hepatology (Baltimore, Md.), 63(3), 1000-1012.

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Multiparameter Flow Cytometry Profiling

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Lymphocytes and myeloid cells were prepared as above. Cells were stained according to standard protocols with combinations of the following anti-mouse antibodies: OX40-APC (eBioscience; clone, OX86), CD4-FITC (eBioscience; clone, GK1.5), CD4-APC-Cy7 (BD Biosciences; clone, GK1.5), CD8–Pacific Orange (Life Technologies; clone, 5H10), CD8–Pacific Blue (BD Biosciences; clone, 53-6.7), TCRb–Pacific Blue (BD Biosciences; clone, H57-597), TCRb-PE (BD Biosciences; clone, H57-597), NK1.1-PE-Cy7 (eBioscience; clone, PK136), CD19–Alexa Fluor 700 (eBioscience; clone, ebio1D3), CD19-APCCy7 (BD Biosciences; clone, 1D3), CXCR5-biotin (BD Biosciences; clone, 2G8), OX40L-PE (eBioscience; clone, RM134L), F4/80-PE-Cy7 (eBioscience; clone, BM8), CD11b-PerCP-Cy5.5 (BD Biosciences; clone, M1/70), CD11c-APC (eBiosciences; clone, N418), major histocompatibility complex II–efluor 450(I-A/I-E) (eBioscience; clone, M5/114.15.2), Ly6g-PerCP-Cy5.5 (BD Biosciences; clone, 1A8), and Ly6c-AF700 (BD Biosciences, clone: AL-21). When applicable for biotinylated antibodies, cells were stained with secondary QDot 605–conjugated streptavidin (Life Technologies). Cells were analyzed using an LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar) or sorted on an Aria III (BD Biosciences).

Publicover J., Gaggar A., Jespersen J.M., Halac U., Johnson A.J., Goodsell A., Avanesyan L., Nishimura S.L., Holdorf M., Mansfield K.G., Judge J.B., Koshti A., Croft M., Wakil A.E., Rosenthal P., Pai E., Cooper S, & Baron J.L. (2018). An OX40/OX40L interaction directs successful immunity to hepatitis B virus. Science translational medicine, 10(433), eaah5766.

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Aortic Cell Profiling by Flow

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Aortic vessels were prepared for single-cell suspensions, surface-stained with CD45-APC-eFluor 780, NK1.1-PE-Cy7 (eBiosciences, CA, USA), TCR-β V450, CD11b-PE, (BD Biosciences, NJ, USA), and analyzed using flow cytometry to study cellular markers. For a detailed description, see Online Supplement.

Keppeler K., Pesi A., Lange S., Helmstädter J., Strohm L., Ubbens H., Kuntić M., Kuntić I., Mihaliková D., Vujačić-Mirski K., Rosenberger A., Küster L., Frank C., Oelze M., Finger S., Zakrzewska A., Verdu E., Wild J., Karbach S., Wenzel P., Wild P., Leistner D., Münzel T., Daiber A., Schuppan D, & Steven S. (2024). Vascular dysfunction and arterial hypertension in experimental celiac disease are mediated by gut-derived inflammation and oxidative stress. Redox Biology, 70, 103071.

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Isolation and Characterization of Liver Leukocytes

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Single-cell lymph node and spleen suspensions were obtained and stained as previously described(11 (link)). For Single cell liver leukocyte suspension the livers were weighted and minced in RPMI 10% FCS DNAse solution and passed through a 40 μm filter. Cells were washed and separated with a Ficoll (Sigma) gradient and washed with either PBS or HBSS. Samples were analyzed on LSR-II flow cytometers (BD Biosciences). Data were analyzed with FlowJo software (TreeStar, Ashland, OR). Isolation of NK cell subsets by flow cytometric cell sorting was performed with FACS-Aria II (BD Bioscience). Antibodies used: CD3 PE, CD45.1 APC, CD3 PE, GR1-APC, NK1.1 PECy7, PerCP eflour 710 and viability dye eFlour®780 (eBioscience); CXCR6 PE and APC (BD Bioscience); CD49a (BD Pharmingen); CD49b, CD11b PeCy7, CD45.2 PB (BioLegend).

Scholz F., Naik S., Sutterwala F.S, & Kaplan D.H. (2015). Langerhans suppress CD49a+ NK cell mediated skin inflammation. Journal of immunology (Baltimore, Md. : 1950), 195(5), 2335-2342.

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Multiparameter Flow Cytometry of Lung Immune Cells

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The following antibodies were used to identify cellular subsets and intracellular cytokines in whole lung homogenates: from BioLegend (San Diego, CA) CD45-PerCP, CD11c-FITC, Ly6G-AF700, MHC class II-BV421, CD4-FITC, CD8a-APC-Cy7, CD3-Q655, NKG2D-PE, NK1.1-PE-Cy7, IFNγ-PE; from eBioscience, CD11b-APC; from BD Biosciences (San Jose, CA) SiglecF-PE. Samples were obtained using an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software version 10 (Ashland, OR). Cells of interest were identified after exclusion of debris, dead cells and doublets (Figure S2).

Walker K.H., Krishnamoorthy N., Brüggemann T.R., Shay A.E., Serhan C.N, & Levy B.D. (2021). Protectins PCTR1 and PD1 Reduce Viral Load and Lung Inflammation During Respiratory Syncytial Virus Infection in Mice. Frontiers in Immunology, 12, 704427.

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