Ki67 antibodies (Biolegend, San Diego, CA, USA) were used to determine cellular proliferation by flow cytometry according to the manufacturer's protocol. Cells (50,000) were acquired using a FACSCalibur (BD, Franklin Lakes, NJ, USA) flow cytometer, followed by analysis using FloJo software (Tree Star, Inc., Ashland, OR, USA).
Ki67 antibody
Manufactured by BioLegend
Sourced in United States
The Ki67 antibody is a laboratory reagent used in various techniques, such as immunohistochemistry and flow cytometry, to detect the presence and localization of the Ki67 protein. The Ki67 protein is a cellular marker associated with cell proliferation and is commonly used to assess the proliferative state of cells.
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Lab products found in correlation
Golgiplug, by BD (2 mentions)
Fitc annexin 5 apoptosis detection kit with 7 aad, by BioLegend (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Fortessa, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Foxp3 fix perm kit, by BioLegend (2 mentions)
Facscalibur, by BD (1 mentions)
Flojo software, by Tree Star (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm reagent kit, by BD (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Tnf α, by BioLegend (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Fortessa x20 flow cytometer, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Live dead kit, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
7 protocols using ki67 antibody
1
Ki67 Expression Analysis by Flow Cytometry
2
Cell Proliferation and Apoptosis Assay
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Cells were seeded in six‐well plates at 100,000 cells/well on the day before drug administration. After treatment for 24 h, attached tumor cells were digested and washed in PBS two to three times. Cells were then fixed and permeated using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Invitrogen, Waltham, MA, USA) overnight and stained with Ki‐67 antibodies (Biolegend, San Diego, CA, USA) for 30 min. In an apoptosis assay, cells were incubated with Annexin V‐PE and 7‐AAD after digestion and washing and incubated in the dark for 10 min. Finally, the stained cells were tested using Cytek NL‐CLC. Results were analyzed using Flowjo software (Flowjo, Ashland, OR, USA).
3
Characterization of Immune Cell Populations
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Freshly isolated cells were used for the procedures. Information on antibodies is listed in Key resources Table . Following markers were used to identify the specific population of cells; total leukocytes (CD45+), neutrophils (Ly6G+), monocytes/macrophages in the gut (CD64+Ly6G−Ly6C+), splenic F4/80hi macrophage (CD11b+F4/80hiLy6G−Ly6C+), splenic monocytes (CD11b+Ly6G−Ly6Chi), and dendritic cells (DC) (CD11c+F4/80−CD64−). The gating strategy for the SI is shown in Fig. S3A . For intracellular cytokines staining (ICS), cells were treated with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml, Sigma Aldrich) and Ionomycin (1 μg/ml, Sigma Aldrich) for 5-6 hrs with GolgiPlug (BD Biosciences) in the last 4 hrs. Cytofix/Cytoperm kit (BD Biosciences) was used for ICS. FOXP3 Fix/Perm Kit (BioLegend) was used for Foxp3 staining. Apoptotic cells were analyzed using FITC-Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend) as recommended by the manufacturer. Cell proliferation was detected by Ki67 staining, where surface-stained cells were fixed and permeabilized using 70% ethanol for 1hr at −20°C followed by staining with Ki67 antibody (16A8, BioLegend). Flow cytometry analysis was performed with BD Fortessa (BD Biosciences), and data were analyzed with FlowJo software (Treestar).
4
Analyzing H295R Cells after BM-hMSC Secretome Treatment
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After treatment with the BM-hMSC secretome or basal media control, H295R cells were analyzed by FACS for proliferation, apoptosis, and inflammatory markers using antibodies against Ki67 antibody (BioLegend, Cat no. 350514), Annexin-V (BioLegend, Cat no. 640919), IL-1β (R&D Systems, Cat no. IC8406A), and TNF-α (BioLegend, Cat no. 502943). In brief, treated cell pellets were harvested and fixed/permeabilized with BD cytofix/cytoperm kit reagent (BD Bioscience, CA, USA) for intracellular staining, per the manufacturer’s instructions. After centrifugation at 1500 rpm for 5 min, a total of 1 × 106 cells were resuspended in 200 μl of antibody solution and incubated for 30 min at room temperature in the dark. After washing, the cells were resuspended in PBS with 2% FBS (v/v) for FACS analysis using (BD, Gallios, Flow-cytometer). Data were analyzed using FlowJo software.
5
Characterization of Immune Cell Populations
6
Multiparametric Flow Cytometry Analysis
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Request a detailed protocol Cells were harvested with Accutase (Sigma-Aldrich), rinsed with PBS, and collected by centrifugation at 500 x g for 5 min, then stained with the LIVE/DEAD kit (Fisher Scientific) according to the manufacturer's instructions. Cells were counted and seeded into a 96-well plate at 3x10 5 cells/well, stained with 100 μl fluorescently labelled antibodies (anti β1, β2, β3-integrin, Biolegend) for 30 min at 4 °C, then washed twice in PBS. For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions, and stained with 100 μl of Ki-67 antibody (Biolegend) for 30 min at 4 °C, then rinsed twice with PBS.
For cell cycle experiments, 1x10 6 cells were collected and fixed in ice-cold 70% ethanol for 30 min at 4 °C. Cells were then stained with propidium iodide (Thermo Fisher) for 15 min. All data were acquired using a BD Fortessa X20 flow cytometer (BD Biosciences), driven by BD FACS Diva software, and analysed using FlowJo (Tree Star, USA).
For cell cycle experiments, 1x10 6 cells were collected and fixed in ice-cold 70% ethanol for 30 min at 4 °C. Cells were then stained with propidium iodide (Thermo Fisher) for 15 min. All data were acquired using a BD Fortessa X20 flow cytometer (BD Biosciences), driven by BD FACS Diva software, and analysed using FlowJo (Tree Star, USA).
7
Cell Viability and Proliferation Assay
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Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) was performed as the manufacturer's protocol. In brief, cells were plated at 1000 cells per well in 96-well plates and cultured at 37°C. After transfection of siRNA, CCK-8 solution was added into the cell culture medium at indicated time. The absorbance at 450 nm was recorded using a microplate reader (Thermo Fisher Scienti c, USA). Ki-67 ow cytometry analysis was applied after 48h transfection. The transfected cells were collected by trypsinization, and incubated with Ki-67 antibody (Biolegend, USA) in dark for 30min. Analysis was performed by using a ow cytometer (DxP Athena, Cytexbio).
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