Anti ki67 sola15

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-Ki67 (SolA15) is a monoclonal antibody used for the detection of the Ki67 protein, a cellular marker associated with cell proliferation. This antibody can be utilized in various immunohistochemical applications to identify and quantify proliferating cells. Its core function is to serve as a tool for researchers and clinicians to study cell growth and division in various biological and clinical contexts.

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Anti-Ki67 (147 citations) Ki67 (SolA15, (34 citations) SolA15 (13 citations) Rat anti-mouse Ki67 (Clone SolA15) antibody (2 citations) Anti-Ki67 coupled with eF660 (clone SolA15) (2 citations) Anti-Ki67 mAb (SolA15, (2 citations) Anti-Ki67 (clone SolA15, (2 citations)

13 protocols using anti ki67 sola15

Phenotypic Analysis of Engineered T Cells

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Mononuclear cells were stained with fluorophore-conjugated monoclonal antibodies specific to murine CD45 (Ly5), Thy1.1 (OX-7), CD8α (53–6.7), PD-1 (J43), Granzyme B (NGZB), IFNγ (XMG1.2), TNFα (MP6-XT22), CD101 (Moushi101, eBiosciences), Slamf6 (330-AJ), Lag3 (C9B7W, Biolegend), KLRG1 (2F1, Biolegend) at a 1:100 dilution in PBS+2.5% FBS. Antibodies were purchased from BD Biosciences unless otherwise noted. To measure intracellular cytokine production, engineered T cells ± Msln_406-414 peptide were incubated in vitro for 5 hours in the presence of GolgiPlug (BD Biosciences), stained for surface antigens, fixed and permeabilized (BD Biosciences Fixation/Permeabilization kit), and then stained with appropriate antibodies. For transcription factor analysis, cells were fixed using Foxp3 staining kit (Tonbo) for 30 min at 4°C, and stained with anti-Tox (TXRX10, Invitrogen) and anti-Tcf1 (C63D9, Cell signaling) at a 1:50 dilution in PBS+2.5% FBS overnight, and washed with perm/wash buffer. To quantify the frequency of cells proliferating, cells were surface stained as above followed by eBioscience Foxp3 Fixation/Permeabilization solutions prior to nuclear staining with anti-Ki67 (SolA15). Data were acquired on an LSRII, FacsCanto, or Fortessa and analyzed using FlowJo V.10.3 (BD Biosciences). Cell numbers infiltrating tissues were normalized to tumor weight.

Stromnes I.M., Hulbert A., Rollins M.R., Basom R.S., Delrow J., Bonson P., Burrack A.L., Hingorani S.R, & Greenberg P.D. (2022). Insufficiency of compound immune checkpoint blockade to overcome engineered T cell exhaustion in pancreatic cancer. Journal for Immunotherapy of Cancer, 10(2), e003525.

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Comprehensive Multiparametric Analysis of Mouse Immune Cells

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Single-cell suspensions were prepared from mouse spleen and pooled lymph nodes (cervical, inguinal, mesenteric, axillary and brachial). For intracellular cytokine staining, lymphocytes were plated at 5×10⁵ cells/well in 96 well tissue culture plates in complete RPMI containing phorbol myristate acetate (50 ng/mL; Sigma-Aldrich), ionomycin (250 ng/mL; Sigma-Aldrich) and monensin (1/1500; BD, Bioscience) for 4 hours at 37°C. All cells were fixed with BD Cytofix™ (BD, Biosciences) or fixed and permeabilized with the eBioscience Foxp3 staining kit (eBioscience). Anti-murine antibodies included anti-CD4 (RM4–5), anti-CD8a (53–6.7), anti-FoxP3 (FJK-16s), anti-CD25 (PC61.5), anti-CD127 (A7R34), anti-CTLA4 (UC10–4B9), anti-CD69 (H1.2F3), anti-CD103 (2E7), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-IL-2 (JES6–5H4), anti-IFNγ (XMG1.2), anti-KLRG1 (2F1), anti-PD1 (J43 and anti-phospho-STAT5 (SRBCZX) and anti-Ki67 (SolA15) from eBioscience. Data were collected on BD CANTO II (BD Biosciences) and analyzed using FlowJo for Mac version 9.6 (Tree Star Inc.).

Humblet-Baron S., Franckaert D., Dooley J., Ailal F., Bousfiha A., Deswarte C., Oleaga-Quintas C., Casanova J.L., Bustamante J, & Liston A. (2018). IFN-γ and CD25 drive distinct pathological features during hemophagocytic lymphohistiocytosis. The Journal of allergy and clinical immunology, 143(6), 2215-2226.e7.

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Multiparameter flow cytometry analysis

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The following monoclonal antibodies were used for multiparameter flow cytometry: anti-CD4 (RM4-5, Biolegend), anti-CD25 (PC61.5, eBioscience), anti-CCR6 (29-2L17, Biolegend), anti-CD103 (2E7, Biolegend), anti-Ly5.1 (A20, Biolegend), anti-Ly5.2 (104, Biolegend), anti-CTLA4 (UC10-4B9, eBioscience), anti-Ki67 (solA15, eBioscience), anti-Bcl-2 (BCL10C4, Biolegend), anti-ICOS (7E.17G, eBioscience), anti-GITR (DTA-1, eBioscience), anti-Helios (22F6, Biolegend), anti-Neuropilin (FAB566A, R&D systems), anti-PD1 (J43, eBioscience), anti-CXCR5 (2G8, BD Biosciences). The Foxp3 staining kit was used for staining for CTLA-4, Ki67, Helios, Bcl-2 and Bcl6. Stained cells were analyzed on FACS Canto (Becton Dickinson) and were sorted on FACS Aria (Becton Dickinson). Data were analyzed with Flowjo software.

Chandrasekaran U., Yi W., Gupta S., Weng C.H., Giannopoulou E., Chinenov Y., Jessberger R., Weaver C.T., Bhagat G, & Pernis A.B. (2016). Regulation of effector Tregs in murine lupus. Arthritis & rheumatology (Hoboken, N.J.), 68(6), 1454-1466.

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Multiparameter Flow Cytometry Analysis

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The antibodies used for cell staining were purchased from BD Biosciences, BioLegend or eBioscience, unless otherwise noted. The following antibodies were used for cell surface staining: anti-CD4 (GK1.5); anti-CD8 (53–6.7); anti-CD43 (1B11); anti-Thy1.1 (OX-7); anti-CD45.1 (A20); anti-Vβ5 (MR9-4); anti-TNFα (MP6-XT22); anti-TNFR1 (55R-286); anti-TNFR2 (TR7554) (R&D Systems). Intracellular Foxp3 staining was performed according to the manufacturer’s recommendation using anti-Foxp3 (FJK-16s) and the Foxp3 staining kit from eBioscience. For intracellular staining of Ki67, cells were surfaced stained before fixation and permeabilized using reagents from the Foxp3 kit (eBioscience) and then stained with anti-Ki67 (SolA15). For intracellular GzmB staining the Cytofix/Cytoperm intracellular staining kit from BD was used according to the manufacturer’s recommendations. Dead cells were excluded by singlet gating strategy and Fixable Viability Dye (eBioscience). The flow cytometric data was collected with an LSRII (BD Biosciences) and analyzed using FlowJo (Tree Star) software.

Joedicke J.J., Myers L., Carmody A.B., Messer R.J., Wajant H., Lang K.S., Lang P.A., Mak T.W., Hasenkrug K.J, & Dittmer U. (2014). Activated CD8+ T cells induce expansion of Vβ5+ regulatory T cells via TNFR2 signaling. Journal of immunology (Baltimore, Md. : 1950), 193(6), 2952-2960.

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Skin Biopsy and Cell Staining

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Biopsies of 0.6 cm in diameter were taken of the injection site, and skin was mechanically disrupted using a sterile scalpel in PBS containing 2 mM EDTA. The resulting cell suspension was stained in 0.5% FBS/PBS 2 mM EDTA using anti-B220 (RA3-6B2, BD Pharmingen), anti-Cd11c (N418, eBiosciences), anti-Cd45 (30-F11, BD Pharmingen), anti-Pdca1 (927, BioLegend), anti-Cd31 (390, eBiosciences), anti-Ki67 (SolA15, eBiosciences), and anti-BrdU (Bu20A, eBiosciences).

Mylonas A., Hawerkamp H.C., Wang Y., Chen J., Messina F., Demaria O., Meller S., Homey B., Di Domizio J., Mazzolai L., Hovnanian A., Gilliet M, & Conrad C. (2023). Type I IFNs link skin-associated dysbiotic commensal bacteria to pathogenic inflammation and angiogenesis in rosacea. JCI Insight, 8(4), e151846.

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Proliferation Analysis of Immune Cells

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Mice were injected i. p. with 500 μg of the nucleoside analog EdU (Baseclick GmbH) in PBS twice, i.e., 24 h and 3 h before euthanizing. After, PPs were prepared for single cell suspension and stained for surface antigens as described, followed by EdU detection using the EdU FC Kit 647 (Baseclick GmbH) according to the manufacturer’s protocol, with anti-Ki67(SolA15, eBioscience) staining afterwards.

Liu H.Y., Giraud A., Seignez C., Ahl D., Guo F., Sedin J., Walden T., Oh J.H., van Pijkeren J.P., Holm L., Roos S., Bertilsson S, & Phillipson M. (2021). Distinct B cell subsets in Peyer’s patches convey probiotic effects by Limosilactobacillus reuteri. Microbiome, 9, 198.

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Multiparametric Flow Cytometry for Hematopoietic Stem Cell Analysis

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Anti-CD127 (A7R34), anti-CD34 (RAM34), anti-c-Kit (2B8), anti-Sca-1 (D7), anti-CD16/32 (93), lineage cocktail (consisting of anti-CD3, anti-B220, anti-CD11b, anti-Ter119, and anti-Gr1, 88-7772), anti-CD150 (mShad150), anti-CD48 (HM48-1), anti-NK1.1 (PK136), anti-CD11b (M1/70), anti-CD3 (17A2), anti-CD19 (eBio1D3 (1D3)), anti-BrdU (BU20A) and anti-Ki67 (SolA15) were purchased from eBioscience. Anti-active caspase 3 (550821) was purchased from BD Bioscience. Antibodies against Myc (9E10) and GST (1-109) were purchased from Santa Cruz Biotechnology. Antibodies against Flag-tag (M1), β-actin (SP124), anti-Zfp90 (SAB2103688) and His-tag (6AT18) were obtained from Sigma-Aldrich. Anti-Snf2l (PA5-41440) and anti-Bptf (730026) were purchased from Invitrogen Antibodies. Antibodies conjugated with Alexa-488 (A11008) or Alexa-594 (A11012) was purchased from Molecular probes Inc. Hoechst 33342 (14533) were purchased from Sigma-Aldrich. Annexin V-FITC/PI apoptosis detection kit (BMS500FI) was purchased from eBioscience.

Liu T., Kong W.X., Tang X.Y., Xu M., Wang Q.H., Zhang B., Hu L.D, & Chen H. (2018). The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells. Cell Death & Disease, 9(6), 677.

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Multiparametric Flow Cytometry Analysis

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Mononuclear cells were isolated by a Percoll gradient (70/40). Isolated cells
were either stained immediately or re-stimulated for T cell cytokine analysis with 50
ng/mL PMA and 750 ng/mL Ionomycin in the presence of Golgi Plug (BD Biosciences, San Jose,
CA) for 4 hours. Single-cell suspensions from spinal cord or spleen cells were incubated
with Fc Block (2.4G2) and the following anti-bodies were used as appropriate: anti-CD4
(RM4-5) (BD Biosciences); anti-Foxp3 (FJK-16s), anti-IFN-γ (XMG1.2), anti-IL-17A
(eBio17B7), and anti-Ki-67 (SolA15) all from eBioscience, San Diego, CA. Cell viability
was determined by staining with Live/Dead Fixable Near-IR Dead Cell Stain Kit (Life
Technologies, Grand Island, NY). To evaluate system x_c⁻expression on immune cells, cells were incubated with Fc Block (2.4G2), then surface
stained using anti-CD11b (M1/70) and CD45 (30-F11) (all from eBioscience) followed by
intracellular staining for system x_c⁻ (Abcam, ab37185). Cell
viability was again determined by staining with Live/Dead Fixable Near-IR Dead Cell Stain
Kit (Life Technologies). Stained cells were run on an LSR II Flow Cytometer (BD
Biosciences) and analyzed by FlowJo (Treestar).

Evonuk K.S., Baker B.J., Doyle R.E., Moseley C.E., Sestero C.M., Johnston B.P., De Sarno P., Tang A., Gembitsky I., Hewett S.J., Weaver C.T., Raman C, & DeSilva T.M. (2015). Inhibition of system xc− transporter attenuates autoimmune inflammatory demyelination. Journal of immunology (Baltimore, Md. : 1950), 195(2), 450-463.

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Multiparametric Phenotyping of Murine T Cells

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For staining of cells directly ex vivo, single cell suspensions of PLN cells and splenocytes were prepared and red blood cells lysed using ACK lysis buffer (Gibco, USA). To distinguish live from dead cells, a Fixable Viability Dye (eBioscience, US) was used. Fc receptors were blocked with anti-CD16/CD32 antibodies for 5 minutes at 4° C before antibody staining was started. The following antibodies were used for phenotyping of murine T cells: anti-CD4 (RM4-5), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-Foxp3 (FJK-16s), and anti-Ki67 (SolA15) (eBioscience, Biolegend or BD Pharmingen, US). For tetramer-stained samples, anti-CD11b (M1/70), anti-CD11c (N418) anti-B220 (RA3-6B2) antibodies (all eBioscience) were added to establish a “dump gate”, excluding irrelevant cells and reducing noise. Extracellular staining was performed by incubating with antibodies for 15–30 minutes at 4°C. All antibodies were used at a 1/100 dilution, except anti-CD44 (1/400). For intracellular staining with anti-Ki67 or -Foxp3, cells were fixed and permeabilized with a Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instructions.

Vazquez-Mateo C., Collins J., Goldberg S.J., Lawson M., Hernandez-Escalante J, & Dooms H. (2019). Combining anti-IL-7Rα antibodies with autoantigen-specific immunotherapy enhances non-specific cytokine production but fails to prevent Type 1 Diabetes. PLoS ONE, 14(3), e0214379.

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Immunofluorescence Imaging of Mouse Ear Pinnae

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Ear pinnae were split with forceps, fixed in 1% paraformaldehyde solution (Electron Microscopy Sciences) overnight at 4°C and blocked in 1% BSA, 0.25% Triton X blocking buffer for 2 hours at room temperature. Tissues were first stained with anti-CD8α (clone 53-6.7, eBioscience), anti-Sca-1 (D7, eBioscience) and/or rabbit anti-CD31 (390, eBioscience), and anti-Ki67 (solA15, eBioscience) antibodies overnight at 4°C, washed three time with PBS and then stained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 5 min at room temperature and before being mounted with ProLong Gold (Molecular Probes) antifade reagent. Ear pinnae images were captured on a Leica TCS SP8 confocal microscope with a 40X oil objective (HC PL APO 40X/1.3 oil). Images were analyzed using Imaris Bitplane software.

Linehan J.L., Harrison O.J., Han S.J., Byrd A.L., Vujkovic-Cvijin I., Villarino A.V., Sen S.K., Shaik J., Smelkinson M., Tamoutounour S., Collins N., Bouladoux N., Dzutsev A., Rosshart S.P., Arbuckle J.H., Wang C.R., Kristie T.M., Rehermann B., Trinchieri G., Brenchley J.M., O’Shea J.J, & Belkaid Y. (2018). Non-classical immunity controls microbiota impact on skin immunity and tissue repair. Cell, 172(4), 784-796.e18.

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