Ki67 alexa fluor 488

Manufactured by BD

Ki67-Alexa Fluor 488 is a fluorescently-labeled antibody that binds to the Ki67 protein, a cellular marker of proliferation. This reagent can be used in flow cytometry and immunofluorescence applications to detect and quantify proliferating cells.

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Lab products found in correlation

Cd45.2 alexafluor700, by Thermo Fisher Scientific (3 mentions) Granzyme b pe texas red, by BD (3 mentions) Cd3 pe cy7, by Thermo Fisher Scientific (3 mentions) Cd8 percp efluor 710, by Thermo Fisher Scientific (2 mentions) Foxp3 alexa fluor 700, by Thermo Fisher Scientific (2 mentions) Granzyme b apc, by BD (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Anti pd 1 clone rmp1 14, by BioXCell (1 mentions) Anti ctla4 9h10, by BioXCell (1 mentions) Cd11b apc efluor780, by Thermo Fisher Scientific (1 mentions) Foxp3 apc, by Thermo Fisher Scientific (1 mentions) Annexin 5, by BioLegend (1 mentions) Triton x 100, by Merck Group (1 mentions) Foxp3 pe, by BioLegend (1 mentions) Cd4 percp cy5, by BioLegend (1 mentions) Il 10 alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Ifn γ alexa fluor 488, by BD (1 mentions) Cd3 pe cy7, by BioLegend (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Isotype control antibody, by BioXCell (1 mentions)

5 protocols using ki67 alexa fluor 488

Anti-PD-1 and Anti-CTLA4 Antibody Protocol

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Therapeutic anti-PD-1 (clone RMP1-14) and anti-CTLA4 (9H10) monoclonal antibodies were produced by BioXcell. Antibodies used for flow cytometry were purchased from the following sources (dilutions are indicated in parentheses): eBioscience (CD45.2 Alexa Fluor 700, cat: 56-0454 (1:200), CD3 PE-Cy7, cat: 25-0031 (1:200), CD4 ef450, cat: 48-0041 (1:200), CD8 PerCP-efluor710, cat: 46-0083 (1:200), CD11b APC-efluor 780, cat: 47-0112 (1:600), ICOS PE, cat: 12-5985 (1:200), PD-L1 PE Cy7, cat: 25-5982-82 (1:200), FoxP3 Alexa Fluor 700, cat: 56-5773 (1:100), FoxP3 APC, cat: 17-5773 (1:200), PD-1 PE-Cy7, cat: 25-9985 (1:200)), Invitrogen (Granzyme B PE-Texas Red, cat: GRB17 (1:125)) and BD Pharmingen (Ki67-Alexa Fluor 488, cat: 561165 (1:50)).

Oseledchyk A., Ricca J.M., Gigoux M., Ko B., Redelman-Sidi G., Walther T., Liu C., Iyer G., Merghoub T., Wolchok J.D, & Zamarin D. (2018). Lysis-independent potentiation of immune checkpoint blockade by oncolytic virus. Oncotarget, 9(47), 28702-28716.

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Cell Cycle and Apoptosis Analysis

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For cell cycle analysis, fully surface stained cells (stained as described above, for staining panel see Table S1) were fixed with 1% formaldehyde (Thermo Fisher Scientific), permeabilized with 0.05% Triton X-100 (Sigma) and subsequently stained with Ki67 AlexaFluor488 (BD Biosciences) and DAPI (Thermo Fisher Scientific). For analysis of apoptosis, cells were surface stained (for antibody panel see Table S1) and subsequently stained with Annexin V (Biolegend).

Peña-Pérez L., Kharazi S., Frengen N., Krstic A., Bouderlique T., Hauenstein J., He M., Somuncular E., Li Wang X., Dahlberg C., Gustafsson C., Johansson A.S., Walfridsson J., Kadri N., Woll P., Kierczak M., Qian H., Westerberg L., Luc S, & Månsson R. (2022). FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors. Frontiers in Immunology, 13, 880668.

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Multiparameter Intracellular Flow Cytometry

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For intracellular staining of Foxp3, ki67, and cytokines, single cell suspensions were fixed and permeabilized using the Foxp3/Transcription factor staining buffer set (eBioscience) according to the manufacturer’s protocol. Cells were stained for 30 minutes at 4 °C with the following directly conjugated antibodies: CD3-PE/CY7, CD4-PerCP/Cy5.5, Foxp3-PE (BioLegend), ki67-AlexaFluor488, IFNγ-AlexaFluor488 (BD Biosciences), IL10-AlexaFluor488 (eBioscience), and Phospho-S6-AlexaFluor488 (Cell Signaling Technology). In addition, anti-mouse CD11b-AlexaFluor488 (BioLegend) was applied to the assessment of retinal macrophages. Acquisition and analysis of cells sorting data were performed on BD FACSCanto II using FlowJo software (Tree Star).

Chen G., Tang L., Wei W., Li Z., Li Y., Duan X, & Chen H. (2016). mTOR regulates neuroprotective effect of immunized CD4+Foxp3+ T cells in optic nerve ischemia. Scientific Reports, 6, 37805.

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Antibody-mediated Immune Profiling

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Therapeutic anti-CTLA-4 (clone 9H10) antibody and isotype control antibody was purchased from BioXcell (cat: BE0131 and BE0087). Antibodies used for flow cytometry were purchased from the following sources (dilutions are indicated in parentheses): eBioscience (CD45.2 Alexa Fluor 700, cat: 56-0454 (1:200), CD3 PE-Cy7, cat: 25-0031 (1:200), CD4 ef450, cat: 48-0041 (1:200), CD4 APC-efluor780, cat: 47-0041 (1:400), CD8 PerCP-efluor710, cat: 46-0083 (1:200), CD11b APC-efluor 780, cat: 47-0112 (1:600), ICOS PE, cat: 12-5985 (1:200), ICOSL PE, cat: 12-5985 (1:200), CTLA-4 PE, cat: 12-1522 (1:200), NK1.1 PE, cat: 12-5941 (1:200), IFNγ PE, cat: 12-7311 (1:200), FoxP3 Alexa Fluor 700, cat: 56-5773 (1:100), FoxP3 APC, cat: 17-5773 (1:200), GATA-3 PE, cat: 12-9966 (1:100), RORγT PerCP-efluor710, cat: 46-6981 (1:100), Tbet PE-Cy7, cat: 25-5825 (1:100), EOMES efluor 450, cat: 48-4875 (1:100), PD-1 PE-Cy7, cat: 25-9985, (1:200)), Biolegend (CD3 BV570, cat: 100225 (1:100), CD11b BV570, cat: 101233 (1:50), Bcl-6 Alexa 594, cat: 648308 (1:50)), Invitrogen (Granzyme B PE-Texas Red, cat: GRB17 (1:125), Granzyme B APC, cat: GRB05 (1:125)) and BD Pharmingen (Ki-67-Alexa Fluor 488, cat: 561165 (1:50), CXCR5-biotin, cat: 551960 (1:100)).

Zamarin D., Holmgaard R.B., Ricca J., Plitt T., Palese P., Sharma P., Merghoub T., Wolchok J.D, & Allison J.P. (2017). Intratumoral modulation of the inducible co-stimulator ICOS by recombinant oncolytic virus promotes systemic anti-tumour immunity. Nature Communications, 8, 14340.

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Immunotherapeutic Response Evaluation

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The commercial sources for reagents were as follows: CpG oligodeoxynucleotide ODN2216 (Invitrogen); We used the following antibodies. Therapeutic anti-CTLA4 (clone 9H10 and 9D9), anti-PD1 (clone RMP1-14), anti-PD-L1 (clone 10F.9G2) were purchased from BioXcell; Antibodies used for flow cytometry were purchased from eBioscience (CD45.2 Alexa Fluor 700, CD3 PE-Cy7, CD4 APC-efluor780, CD8 PerCP-efluor710, FOXP3 Alexa Fluor 700, MHC Class I APC, CD40 APC, CD80 APC, CD86 APC), Invitrogen (CD4 QDot 605, Granzyme B PE-Texas Red, Granzyme B APC), BD Pharmingen (Ki-67-Alexa Fluor 488).

Dai P., Wang W., Yang N., Serna-Tamayo C., Ricca J.M., Zamarin D., Shuman S., Merghoub T., Wolchok J.D, & Deng L. (2017). Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells. Science immunology, 2(11), eaal1713.

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