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Ki67 pe

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Ki67-PE is a laboratory reagent used in flow cytometry applications. It is a fluorescent-labeled antibody specific for the Ki-67 protein, which is a marker of cellular proliferation. Ki67-PE can be used to detect and quantify the proportion of actively dividing cells in a sample.

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Lab products found in correlation

Permeabilization buffer, by Thermo Fisher Scientific (2 mentions) Brdu flow kit, by BD (2 mentions) Il 17a, by Thermo Fisher Scientific (2 mentions) Intracellular fixation permeabilization buffer, by Thermo Fisher Scientific (2 mentions) Tcrb pecy7, by Thermo Fisher Scientific (2 mentions) Brefeldin a 1000x, by Thermo Fisher Scientific (2 mentions) Live dead fixable blue dead cell stain kit for uv excitation, by Thermo Fisher Scientific (2 mentions) Ghost 780, by Cytek Biosciences (2 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions) Attune nxt, by Thermo Fisher Scientific (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Fortessa, by BD (2 mentions) Cd11b percp cy5, by Thermo Fisher Scientific (2 mentions) Cd45.1 pe, by Thermo Fisher Scientific (2 mentions) Moflow, by Beckman Coulter (2 mentions) Canto 2, by BD (2 mentions) Annexin 5 apc, by Thermo Fisher Scientific (2 mentions) Streptavidin pecy5, by BD (2 mentions) Cd117 pecy7, by Thermo Fisher Scientific (2 mentions) Cd4 pe or pb, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

Anti-mouse Ki67 FITC or PE

11 protocols using ki67 pe

1

Single-cell Immunophenotyping and Sorting

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Single-cell suspensions were prepared as described and incubated with Live/Dead Fixable Blue Dead Cell Stain (ThermoFisher) in PBS for 10 min at room temperature followed by blocking with anti-mouse CD16/CD32 (2.4G2) (UCSF Hybridoma Core Facility) and 5% normal rat serum for 10 min at room temperature. Cells were then washed in FACS buffer and stained for surface markers for 20 min at room temperature. For intracellular staining, cells were fixed and permeabilized using the eBioscience FoxP3 Transcription Factor Buffer Set (ThermoFisher) according to the manufacturer’s instructions. Cells were either sorted directly into DMEM (ThermoFisher) containing 10% FBS using a BD FACS Aria Fusion (BD Biosciences) or analyzed using a BD LSR II (BD Biosciences) housed within the UCSF Single Cell Analysis Center. Flow cytometry data was analyzed using BD FACSDiva v8.0 or FlowJo v10.5.3 software (TreeStar Software). The following antibodies were used in this study: Ly51-PE (6C3, BioLegend), CD11c-PE-Cy7 (N418, eBioscience), CD45-PerCP (30-F11, BioLegend), EPCAM-APC-Cy7 (G8.8, BioLegend), Aire-e660 (5H12, eBioscience), Ki67-PE (eBioscience), CCL21 (59106, R and D Systems), Goat anti-Rat IgG-A488 (ThermoFisher).
Wells K.L., Miller C.N., Gschwind A.R., Wei W., Phipps J.D., Anderson M.S, & Steinmetz L.M. (2020). Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells. eLife, 9, e60188.
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2

Mass Cytometry Validation of Blood Samples

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Samples used for mass cytometry validation were 200 μL of whole blood that were fixed in 1-step Fix/Lyse Solution (eBioscience), and stored at −80 °C. Fixed cells were thawed, permeabilized using Permeabilization Buffer (eBioscience) and then stained with the following antibodies specific for epitopes insensitive to fixation, purchased from Biolegend unless otherwise indicated: CD38 BB515 (HIT2, BD Biosciences), CD4 BV510™ (SK3), CD8 PerCP/Cy5.5 (HIT8a), CD161 APC (HP-3G10, eBioscience), HLA-DR APC/Cy7 (L243), TCR Vα7.2 BV605™ (3C10), TCR γ/δ BV421™ (B1), Ki67 PE (Ki-67), and CD3 BV650™ (UCHT1).
Howson L.J., Napolitani G., Shepherd D., Ghadbane H., Kurupati P., Preciado-Llanes L., Rei M., Dobinson H.C., Gibani M.M., Teng K.W., Newell E.W., Veerapen N., Besra G.S., Pollard A.J, & Cerundolo V. (2018). MAIT cell clonal expansion and TCR repertoire shaping in human volunteers challenged with Salmonella Paratyphi A. Nature Communications, 9, 253.
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3

Cell Proliferation Assay with Ki67

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Murine cells stained with cell surface markers were fixed, permeabilized and refixed using buffers from BD Biosciences BrdU Flow Kit (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. After fixation, cells were stained for 30 mins at RT with Ki67 PE (eBioSciences) and Alexa 488 goat anti-rabbit secondary antibody (Abcam) for 20 mins at RT.
Hormaechea-Agulla D., Matatall K.A., Le D.T., Kain B., Long X., Kus P., Jaksik R., Challen G.A., Kimmel M, & King K.Y. (2021). Chronic Infection drives Dnmt3a-Loss of function Clonal Hematopoiesis via IFNg signaling. Cell stem cell, 28(8), 1428-1442.e6.
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4

Intracellular Cytokine Staining for T Cell Activation

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Intracellular cytokine staining for T cells was conducted on day 3, after the T cells were cultured for 72 h. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 h. Brefeldin A 1000X (eBioscience) was added for the final 4.5 h. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4 °C T cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5, FITC; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17 A (FITC or PE; eBioscience), and/or KI67 (PE; eBioscience). Stained cells were analyzed on Fortessa (BD), Attune NxT (Invitrogen), or Accuri C6 (BD). Data was analyzed using FlowJo software (TreeStar). Flow plots showing percent live cells are showing the percent live cells of total lymphocytes. Flow plots show IL-17 producing cells as percent cytokine producing cells of CD4+TCRβ+ Live cells or TCRβ+ Live cells.
O’Driscoll C.A., Gallo M.E., Fechner J.H., Schauer J.J, & Mezrich J.D. (2019). Real-world PM extracts differentially enhance Th17 differentiation and activate the aryl hydrocarbon receptor (AHR). Toxicology, 414, 14-26.
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5

Intracellular Cytokine Staining for T Cells

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Intracellular cytokine staining for T cells was conducted on day 3, after the T cells were cultured for 72 hours. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 hours. Brefeldin A 1000X (eBioscience) was added for the final 4.5 hours. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4°C. T cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17A (FITC; eBioscience), KI67 (PE; eBioscience), or FOXP3 (eFluor450; eBioscience). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD4+TCRβ+ Live cells or TCRβ+ Live cells.
O’Driscoll C.A., Gallo M.E., Hoffmann E.J., Fechner J.H., Schauer J.J., Bradfield C.A, & Mezrich J.D. (2018). Polycyclic aromatic hydrocarbons (PAHs) present in ambient urban dust drive proinflammatory T cell and dendritic cell responses via the aryl hydrocarbon receptor (AHR) in vitro. PLoS ONE, 13(12), e0209690.
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6

Multi-lineage Hematopoietic Cell Analysis

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Total white blood cells from hematopoietic organs were stained in PBS supplemented with 2% FBS with fluorochrome-conjugated mouse antibodies against specific hematopoietic lineage markers. For the analysis of transplant-receiving mice, WBM was stained with fluorochrome-conjugated mouse antibodies to discriminate cells derived from competitors and donors (CD45.1+ and CD45.2+ respectively) and GFP expression was used to precisely define donor-derived cells (GFP+ CD45.2+). Fluorochrome-conjugated mouse antibodies were obtained from Becton Dickinson (Streptavidin/PeCy5, CD117/PeCy7, CD45.1/PE, CD4/PE or PB, CD8/PeCy7 or APC, CD19/APC, B220/APCCy7, TCRβ/PE, CD279/BV421, CD11b/PerCP-Cy5.5, Annexin V/APC, Ki67/PE) and eBiosciences (CD117/APCCy7, Sca-1/APC, CD45.2/APC, Gr1/PE). Hoescht 33342 was obtained from Invitrogen. APC BrdU Flow kit (Becton Dickinson) was used according to the manufacturer’s instruction. Cell sorting was performed either on a MoFlow (Beckman Coulter) or Influx (Becton Dickinson) cell sorter and analysis on a Canto II (Becton Dickinson). FACS data were analyzed by FlowJo Software (v8.8.7).
Scourzic L., Couronné L., Pedersen M.T., Della Valle V., Diop M., Mylonas E., Calvo J., Mouly E., Lopez C.K., Martin N., Fontenay M., Bender A., Guibert S., Dubreuil P., Dessen P., Droin N., Pflumio F., Weber M., Gaulard P., Helin K., Mercher T, & Bernard O.A. (2016). DNMT3AR882H mutant and Tet2 inactivation cooperate in the deregulation of DNA methylation control to induce lymphoid malignancies in mice. Leukemia, 30(6), 1388-1398.
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7

Multi-lineage Hematopoietic Cell Analysis

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Total white blood cells from hematopoietic organs were stained in PBS supplemented with 2% FBS with fluorochrome-conjugated mouse antibodies against specific hematopoietic lineage markers. For the analysis of transplant-receiving mice, WBM was stained with fluorochrome-conjugated mouse antibodies to discriminate cells derived from competitors and donors (CD45.1+ and CD45.2+ respectively) and GFP expression was used to precisely define donor-derived cells (GFP+ CD45.2+). Fluorochrome-conjugated mouse antibodies were obtained from Becton Dickinson (Streptavidin/PeCy5, CD117/PeCy7, CD45.1/PE, CD4/PE or PB, CD8/PeCy7 or APC, CD19/APC, B220/APCCy7, TCRβ/PE, CD279/BV421, CD11b/PerCP-Cy5.5, Annexin V/APC, Ki67/PE) and eBiosciences (CD117/APCCy7, Sca-1/APC, CD45.2/APC, Gr1/PE). Hoescht 33342 was obtained from Invitrogen. APC BrdU Flow kit (Becton Dickinson) was used according to the manufacturer’s instruction. Cell sorting was performed either on a MoFlow (Beckman Coulter) or Influx (Becton Dickinson) cell sorter and analysis on a Canto II (Becton Dickinson). FACS data were analyzed by FlowJo Software (v8.8.7).
Scourzic L., Couronné L., Pedersen M.T., Della Valle V., Diop M., Mylonas E., Calvo J., Mouly E., Lopez C.K., Martin N., Fontenay M., Bender A., Guibert S., Dubreuil P., Dessen P., Droin N., Pflumio F., Weber M., Gaulard P., Helin K., Mercher T, & Bernard O.A. (2016). DNMT3AR882H mutant and Tet2 inactivation cooperate in the deregulation of DNA methylation control to induce lymphoid malignancies in mice. Leukemia, 30(6), 1388-1398.
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8

Comprehensive T Cell Phenotyping Protocol

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Splenocytes were stained with fixable viability dye eFluor780 (1:1,000) before staining for surface antigens CD4-A700 (GK1.5, 1:100), CD69-PE-Cy7 (H1.2F3, 1:300), CD62L-PerCP-Cy5.5 (MEL-14, 1:200), LAG-3-PE (C9B7W, 1:200), TIM-3-PE (8B.2C12, 1:200) (all eBioscience) and PD-1-PE-Cy7 (29F.1A12, 1:300), TIGIT-APC (1G9, 1:200), CD49b-PE (HMα2, 1:100) (all Biolegend). For staining of transcription factors and Ki67, cells were then fixed with fixation/permeabilization buffer and antibodies were diluted in permeabilization buffer (both eBioscience); Ki67-PE (SolA15, 1:200), c-Maf-eFluor660 (SYMOF1, 1:200), NFIL3-PE (S2M-E19, 1:200), FoxP3-eFluor450 (FJK-16S, 1:100). Data were collected using an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software (Treestar).
Burton B.R., Britton G.J., Fang H., Verhagen J., Smithers B., Sabatos-Peyton C.A., Carney L.J., Gough J., Strobel S, & Wraith D.C. (2014). Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy. Nature Communications, 5, 4741.
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9

BrdU and Ki67 Cell Proliferation Assay

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For cell proliferation studies, 50 mg/kg of BRDU (Sigma) was injected interperitoneally starting at 12 h after reperfusion and again at 24 and 48 h. BRDU staining was assessed using a BRDU Flow Kit (BD Biosciences). In brief, cells were permeabilized with detergent and treated with DNAse prior to the addition of both anti-BRDU-FITC antibody and Ki67-PE (eBioscience).
Ritzel R.M., Patel A.R., Grenier J.M., Crapser J., Verma R., Jellison E.R, & McCullough L.D. (2015). Functional differences between microglia and monocytes after ischemic stroke. Journal of Neuroinflammation, 12, 106.
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10

Multiparameter Flow Cytometry Panel

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Splenocytes were stained with fixable viability dye eFluor780 (1:1,000) before staining for surface antigens CD4-A700 (GK1.5, 1:100), CD69-PE-Cy7 (H1.2F3, 1:300), CD62L-PerCP-Cy5.5 (MEL-14, 1:200), LAG-3- PE (C9B7W, 1:200), TIM-3-PE (8B.2C12, 1:200) (all eBioscience) and PD-1-PE-Cy7 (29F.1A12, 1:300), TIGIT-APC (1G9, 1:200), CD49b-PE (HMα2, 1:100) (all Biolegend). For staining of transcription factors and Ki67, cells were then fixed with Fixation/permeabilisation buffer and antibodies were diluted in permeabilisation buffer (both eBioscience); Ki67-PE (SolA15, 1:200), c-Maf-eFluor660 (SYMOF1, 1:200), NFIL-3-PE (S2M-E19, 1:200), FoxP3-eFluor450 (FJK-16S, 1:100). Data were collected using an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software (Treestar).
Burton B.R., Britton G.J., Fang H., Verhagen J., Smithers B., Sabatos-Peyton C.A., Carney L.J., Gough J., Strobel S, & Wraith D.C. (2014). Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy. Nature Communications, 5, 4741.
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