0.5 × 106 CLBL-1 cells were harvested after 72-hour of DMSO or pevonedistat treatment. Cells first were stained with the fixable viability dye, Ghost Dye Red 780 (1:1000; Tonbo Biosciences, San Diego, CA). For intracellular Ki67 staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA) and further labelled with FITC anti-Ki67 (1:20; BD Biosciences, San Jose, CA). DAPI solution (1:250; Thermo Fisher Scientific, Madison, WI) was then added and incubated overnight at 4°C. Samples were analysed on a BD LSRFortessa (BD Biosciences, San Jose, CA), and results were analysed using FlowJo v10.0.7 software.
Anti ki67 fitc
Manufactured by BD
Sourced in United States
Anti-Ki67 FITC is a fluorescently labeled antibody that binds to the Ki67 protein, which is a marker of cellular proliferation. It can be used in various laboratory techniques, such as flow cytometry and immunohistochemistry, to detect and quantify proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45ra qdot655, by Thermo Fisher Scientific (4 mentions)
Anti cd25 apc, by BD (4 mentions)
Anti cd8 qdot605, by Thermo Fisher Scientific (4 mentions)
Anti foxp3 pe, by Thermo Fisher Scientific (4 mentions)
Anti cd8 percp, by BD (3 mentions)
Anti bcl2 pe, by BD (3 mentions)
Anti cd8 pacblue, by BD (3 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Anti cd3 pacblue, by BD (3 mentions)
Lsrfortessa flow cytometer, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Anti cd4 qdot605, by Thermo Fisher Scientific (3 mentions)
Anti il22 percp efluor710, by Thermo Fisher Scientific (3 mentions)
Ghost dye red 780, by Cytek Biosciences (2 mentions)
Lsrfortessa, by BD (2 mentions)
Dapi solution, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Foxp3 staining buffer, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Anti cd127 pe cy7, by BD (2 mentions)
29 protocols using anti ki67 fitc
1
Evaluating Cell Proliferation by Flow Cytometry
2
JAK Inhibitor Effects on CLBL-1 Cell Proliferation
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After 72‐hour JAK inhibitor treatment, 0.5 × 106 CLBL‐1 cells were harvested. Cells first were stained with the fixable viability dye, Ghost Dye Red 780 (Tonbo Biosciences20 ; 1 : 1000). For intracellular Ki67 staining, cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit (BD Biosciences17 ) and further labeled with FITC anti‐Ki67 (BD Biosciences17 ; 1 : 20). DAPI solution (Thermo Fisher Scientific10 ; 1 : 250) then was added and incubated overnight at 4°C. Samples were analyzed the next day on a BD LSRFortessa (BD Biosciences17 ) and results were analyzed with FlowJo v10.0.7 software18 .
3
Ki67-Hoechst Assay for Cell Cycle Analysis
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For Ki67-Hoechst assays, CD34+ cells with or without editing were first stained with fixable viability stain 660 (1:1000, BD) for 5 min in 37° C and were fixed with Cytofix/Cytoperm buffer (BD) for 15 min in 4° C. Cells were stained with FITC-anti-KI67 (1:25, BD, 556027) for 2hours-overnight in Permwash buffer (BD), then with Hoechst 33342 (1:5000; Life Technologies) for 5 min at RT. Samples were sorted on a Aria Fusion Cell Sorter (BD) or analyzed on a LSR Fortessa cytometer (BD). For assessment of immunophenotypic markers together with cell cycle analysis, human CD34+ cells with or without editing were stained with Percp-Cy5.5-anti-CD34 (1:50) and PE-Cy7-anti-CD38 (1:50) for 30 min in 4° C before they were stained with fixable viability stain and fixed. For assessment of cell cycle status without fixation (live cell cycle status), cells with or without editing were stained with Hoechst 33342 (1:1000, Invitrogen) for 45min in 37° C, and then were stained with Pyronin Y (1:20,000, Invitrogen) for additional 15 min in 37° C or were just stained with Pyronin Y for 15 min in 37° C. Samples were then sorted on Aria Fusion Cell Sorter (BD) or analyzed on LSR Fortessa cytometer (BD).
4
Quantification of CD4+ T cell activation
5
Multiparameter Flow Cytometry of T-cell Subsets
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Purified CD8+ T cells, splenocytes and thymocytes were first stained for surface antigens and then treated with Foxp3 staining buffer set according to the manufacturer's directions (eBioscience). Anti-Eomes AlexaFluor 647 or eFluor 660 (Dan11mag, 1/75), anti-T-bet PE (eBio4B10, 1/100) and anti-CD49d FITC or PE (R1-2, 1/50) antibodies were purchased from eBioscience. Anti-CD8 PercP (53–6.7, 1/50), anti-CXCR3 APC (CXCR3-173, 1/50), anti-CD4 Pe-Cy7 (RM4-5, 1/100), anti-CD62L PE (1/100) or V450 (1/50) (MEL-14), anti-Bcl2 PE (3F11, 1/25), anti-Ki67 FITC (B56, 1/25), anti-CD44 FITC or V450 (IM7, 1/50), anti-CD127 Pe-Cy7 (SB/199, 1/50), anti-CD122 FITC (TM-BETA1, 1/50), anti-NK1.1 FITC (PK136, 1/50), anti-CD90.2 Pe-Cy7 (53-2.1, 1/100) and anti-IFNγ APC or PB or PE (XMG1.21/50) were purchased from BD biosciences. Anti-CD3 Pe-Cy7 (2C11, 1/100) was purchased from Biolegend.
In some experiments, brefeldin A (5 μg ml−1, Sigma) was added in samples for 3 h at 37 °C before intracytoplasmic staining. Blood samples were directly stained for surface antigens and then treated with FACS lysing buffer (BD biosciences) as described in the product data sheet. All samples were fixed with 1% paraformaldehyde in PBS prior to their processing using a Cyan flow cytometer (Dako Cytomation).
In some experiments, brefeldin A (5 μg ml−1, Sigma) was added in samples for 3 h at 37 °C before intracytoplasmic staining. Blood samples were directly stained for surface antigens and then treated with FACS lysing buffer (BD biosciences) as described in the product data sheet. All samples were fixed with 1% paraformaldehyde in PBS prior to their processing using a Cyan flow cytometer (Dako Cytomation).
6
Multiparametric Flow Cytometry Analysis
7
Phenotypic Characterization of Immune Cells
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The following monoclonal antibodies (MAbs) were used for phenotypic analysis: (i) anti-CD27-APC-eFluor780, anti-CD45RA-phycoerythrin (PE), and anti-CD127-eFluor450 (eBioscience, San Diego, CA); (ii) anti-CD3-Pacific Blue, anti-CD8-AmCyan, anti-CD8-V500, anti-CD11a-fluorescein isothiocyanate (FITC), anti-CD95-PE, anti-Ki67-FITC, and anti-CCR7-PE-Cy7 (BD Biosciences, Heidelberg, Germany); (iii) anti-CCR7-FITC (R&D Systems); and (iv) anti-PD-1-PE-Cy7 (BioLegend, San Diego, CA). 7-Aminoactinomycin D (7-AAD; Viaprobe; BD Biosciences) was used for the exclusion of dead cells. All samples were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR).
8
Comprehensive Treg Immunophenotyping
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To determine the Treg profile and function, 1x106 PBMCs were incubated for 15 min at room temperature with the anti-CD3-PerCP-Cy™5.5 (clone UCHT1), anti-CD4-APC-H7 (clone SK3), anti-CD8-Alexa Fluor® 700 (clone RPA-T8), anti-CD25-PE-CF594 (clone M-A251), anti-CD127-Alexa Fluor® 647 (clone HIL-7R-M21), anti-CD45RA-PE-Cy™7 (clone HI100), anti-CD39-BV650 (clone TU66), and anti-CD73-BV421 (clone AD2) monoclonal antibodies (all from BD Biosciences). Cells were then washed and fixed/permeabilized (eBioscience, catalog no. 00-5521-00) using FOXP3 staining buffer (eBioscience, catalog no. 00-5523-00), and finally stained with the combination of conjugated antibodies anti-Ki67-FITC (BD Biosciences, catalog no. 556026) and anti-FOXP3-PE (BD Biosciences, clone 259D/C7) for nuclear epitope staining. Next, cells were washed with PBS, fixed with 1% formaldehyde in PBS (Sigma-Aldrich, catalog no. 1004960700) for 15 min at room temperature and analyzed by flow cytometry.
9
Multiparametric Flow Cytometry for T Cell Profiling
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PBMCs were mixed gently with anti-CD4-FITC and anti-CD25-APC, and anti-CD8-APC antibodies (BD Pharmingen™, USA) respectively and incubated for 30 min at 4 °C, and washed twice. Then, fixed, permeabilized, and stained with anti-Foxp3-PE, anti-IFNγ-PE-CY7, anti-IL4-APC-CY7, anti-IL17-BV421, anti-TNFα-Percpcy5.5, anti-Ki67-FITC, anti-Granzyme B-PE, and anti-TGFβ-APC (BD Pharmingen™, USA) for analysis of T cells. The samples were performed by flow cytometry on a FACS Aric III flow cytometer (BD Biosciences, USA). Flow cytometry data were analyzed using FlowJo software 10.4.
10
Multiparameter Flow Cytometry Immunoassay
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Antibodies used for surface staining included anti-CD14 Qdot 655, anti-CD20 Qdot 655, anti-CD4 PeCy5.5, anti-CD8 Qdot605 (Invitrogen; Carlsbad, California), anti-CD14 BV650, anti-CD20 BV650 (Biolegend; San Diego, California), anti-CD28 ECD (Beckman Coulter; Fullerton, CA), anti-CD25 APC-Cy7, anti-CD95 PE-Cy5, and anti-HLA-DR APC (BD Pharmingen; San Diego, California). Antibodies used for intracellular staining included anti-interleukin-2 (IL-2) Alexa700 (Biolegend), anti-interferon-gamma (IFNγ) Alexa 700 (Invitrogen), anti-tumor necrosis factor-alpha (TNFα) Alexa 700, anti-CD3 Pac Blue, anti-CD69 PE, and anti-Ki67 FITC (BD Pharmingen).
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