Splenocytes were either stimulated with survivin or MUC1 protein for 12 h. Following incubation, the cells were stained with the following surface antibodies: APC anti-mouse CD8a (Biolegend), PE-Cy7 anti-mouse CD4 (Biolegend). Cells were then permeabilized and washed with the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions. Intracellular Ki67 were subsequently stained with PE anti-mouse Ki67 (Biolegend). For central memory T cells (TCM) and effector memory T (TEM) cells, in addition to APC anti-mouse CD8a (Biolegend), PE anti-mouse CD44 (Biolegend) and PE-Cy7 anti-mouse CD62L (Biolegend) were used for cell surface staining. After staining, cells were washed, fixed, and analyzed using flow cytometry.
Pe anti mouse ki67
Manufactured by BioLegend
The PE anti-mouse Ki67 is a fluorescently labeled antibody that binds to the Ki67 protein, a cellular marker of proliferation. It is a tool for flow cytometric analysis of cell proliferation in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Apc anti mouse cd8a, by BioLegend (3 mentions)
Pe cy7 anti mouse cd4, by BioLegend (2 mentions)
Apc anti mouse cd206, by BioLegend (2 mentions)
Pe cy7 anti mouse cd62l, by BioLegend (1 mentions)
Pe anti mouse cd44, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Fitc anti mouse f4 80, by BioLegend (1 mentions)
Apc anti mouse gr 1, by BioLegend (1 mentions)
Mouse regulatory t cell staining kit 1, by Thermo Fisher Scientific (1 mentions)
Pe anti mouse cd11b, by BioLegend (1 mentions)
Liberase, by Roche (1 mentions)
Apc cyanine7 anti mouse cd45, by BioLegend (1 mentions)
Percp anti mouse cd8a, by BioLegend (1 mentions)
Brilliant violet 421 anti mouse cd3, by BioLegend (1 mentions)
Pe cyanine7 anti mouse ifn γ, by BioLegend (1 mentions)
Live dead fixable green dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Fitc anti mouse cd3, by BioLegend (1 mentions)
Pe anti mouse cd86, by BioLegend (1 mentions)
4 protocols using pe anti mouse ki67
1
Multicolor Flow Cytometry Analysis of T Cell Subsets
2
Quantification of Tumor-Associated Immune Cells
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Tumor tissues, excised from euthanized mice, were minced and digested using liberase (Roche) for 2 h. The cells were washed and counted, followed by extracellular staining with antibodies, such as APC anti-mouse Gr-1 (Biolegend), PE anti-mouse CD11b (Biolegend), FITC anti-mouse F4/80 (Biolegend), and APC anti-mouse CD206 (Biolegend), to analyze MDSCs and TAMs in the tumor. For analysis of the tumor-infiltrated proliferative T cells, the cells were washed, surface-stained using PE-Cy7 anti-mouse CD4 (Biolegend), and APC anti-mouse CD8a (Biolegend), fixed, and permeabilized, followed by intracellular staining using PE anti-mouse Ki67 (Biolegend). Cells were washed and analyzed using flow cytometry. Detection of Tregs (regulatory T cells) was performed using Mouse Regulatory T Cell Staining Kit#1 (eBioscience), according to manufacturer’s instructions.
3
Profiling Tumor Immune Cells in OX40 Antibody-Treated Mice
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OX40-humanized mice were implanted with MC38 tumor cells and treated with OX40 antibodies on days 9 and 14 post implantation. On day 17, tumors were collected and dissociated into single cell suspensions by using a digestive solution (1640 medium + 2%FBS + Collagenase IV (Sigma, C5138) + DNase I (Sigma, D5025)), and spleens were ground with sterilized glass slides and filtered through a steel mesh. Red blood cells were lysed using red cell lysing buffer (TIANGEN, RT122). Single cell suspensions were first incubated with the LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Invitrogen, L34970), then labeled with the following antibodies in flow cytometric analyses: Brilliant Violet 421™ anti-mouse CD3 (BioLegend, 100228), PE anti-mouse Ki-67 (BioLegend, 652404), PerCP anti-mouse CD8a (BioLegend, 100732), APC/Cyanine7 anti-mouse CD45 (BioLegend, 103116), PE/Cyanine7 anti-mouse IFN-γ (BioLegend, 505826), eFluor™ 450 anti-FoxP3 (Invitrogen, 48-5773-82) and eFluor™ 506 anti-CD4 (Invitrogen, 69-0042-82). Intracellular FoxP3, IFN-γ and Ki67 were labeled following the product manual. Flow cytometry analysis was performed using Cytek® Aurora (Cytek Biosciences).
4
Tumor Immune Profiling via Single-Cell Flow Cytometry
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Single-cell suspensions from murine tumor tissues were prepared by mechanical and enzymatic disruption in PBS with 1 mg/ml collagenase D and 4 μg/ml DNase I for 1 h in 37 °C with periodic vortexing. Cells were filtered through a 70-um cell strainer, resuspended in PBS and centrifuged at 500 g for 5 min, followed by the removement of the red blood cells (RBCs) with RBC lysis buffer. The cells were blocked by 0.5% BSA for 30 min on ice with FC block and then stained with the following antibodies separately: PerCP/Cyanine5.5 anti-mouse CD45 (103,131; BioLegend), APC/Cyanine7 anti-mouse F4/80 (123,117; BioLegend), FITC anti-mouse CD3 (100,203; BioLegend), PE anti-mouse CD69 (104,507; BioLegend), APC anti-mouse CD206 (141,707; BioLegend), PE anti-mouse CD86 (105,007; BioLegend). APC anti-mouse CD8a (100,711; BioLegend). PE anti-mouse IFN-γ (505,807; BioLegend), PE anti-mouse PD-1 (135,205; BioLegend) and PE anti-mouse Ki67 (652,403; BioLegend). For intracellular staining, cells were first fixed (eBioscience, IC fixation buffer) and permeabilized (eBioscience, 1 × permeabilization buffer).
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