Brilliant stain buffer

Manufactured by Thermo Fisher Scientific

Brilliant stain buffer is a specialized reagent used in various laboratory techniques. It is designed to prepare and stabilize samples for analysis, ensuring consistent and reliable results.

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5 protocols using brilliant stain buffer

Cell Cycle Analysis by FACS

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Cells were collected by trypsinization, resuspended in ice-cold PBS, and fixed by adding ice-cold ethanol. After 20 minutes of incubation, cells were centrifuged for 10 minutes at 4°C and 244g, resuspended in 0.5 mL PBS/RNase solution containing 50 μg/mL DAPI for 20 minutes in the dark and analyzed by FACS. To determine the percentage of Ki67^lo cells in G0-G1 phase of the cell cycle, cells were fixed in ethanol and stained in 100 μL of Brilliant Stain Buffer (BD Horizon) with anti-Ki67 for 30 minutes in the dark. After 2 washes in the Brilliant Stain Buffer, cells were resuspended in regular medium, stained with Vybrant DyeCycle Ruby (Invitrogen), and analyzed by FACS by Bio-Rad ZE5 #2 Cell Analyzer (Bio-Rad) at the UM Flow Cytometry Core.

Augimeri G., Gonzalez M.E., Paolì A., Eido A., Choi Y., Burman B., Djomehri S., Karthikeyan S.K., Varambally S., Buschhaus J.M., Chen Y.C., Mauro L., Bonofiglio D., Nesvizhskii A.I., Luker G.D., Andò S., Yoon E, & Kleer C.G. (2023). A hybrid breast cancer/mesenchymal stem cell population enhances chemoresistance and metastasis. JCI Insight, 8(18), e164216.

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Comprehensive Immune Profiling of Tumor Samples

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Tumors were collected, minced, and digested with collagenase B (0.5 mg/mL, Roche) and hyaluronidase (0.5 mg/mL, Absin) at 37°C for 1 hour. Suspensions were filtered through 40 µm cell strainers. Then, cells were suspended and stained with Fixable Viability Stain 700 (BD Biosciences). Cells were stained according to the standard protocol for flow cytometry. The following antibodies and buffers were used (all reagents from BD Biosciences unless otherwise indicated): anti-CD45 (560510), anti-CD3e (562600), anti-CD8α (563068), anti-CD25 (553075), anti-CD69 (566500), anti-Ki67 (556027), anti-TNF-α (563943), anti-IFN-γ (560660), anti-Perforin (ThermoFisher, 11-9392-82), anti-Granzyme-B (BioLegend, 372204), anti-CD11c (566504), anti-I-A/I-E (BioLegend, 107608), anti-CD80 (560016), anti-CD86 (561962), Brilliant Stain Buffer (563794), and FOXP3/Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00). Cells per 100 mg tumor were counted by Beckman Vi-Cell Auto. Flow cytometry was performed with Beckman CytoFLEX LX.

Yi M., Wu Y., Niu M., Zhu S., Zhang J., Yan Y., Zhou P., Dai Z, & Wu K. (2022). Anti-TGF-β/PD-L1 bispecific antibody promotes T cell infiltration and exhibits enhanced antitumor activity in triple-negative breast cancer. Journal for Immunotherapy of Cancer, 10(12), e005543.

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Comprehensive Immune Profiling Protocol

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Anti (α)-mouse CTLA-4, α-mouse programmed death-ligand 1 (PD-L1), and mouse IgG2b isotype antibodies were purchased from BioXCell (West Lebanon, New Hampshire, USA). Mouse Ki67 Alexa Fluor 647, PD-1 PE, GR-1(Ly6G/Ly6C) Brilliant Violet 650, DX5 Dazzle 594, CD11c PE/Cy7, LAG3 Brilliant Violet 650, TIM3 PE/Cy7, CD3e PerCP/Cy5.5, CD11b FITC, CD45 APC/Fire 750, α-CD4 Brilliant Violet 421 and α-CD8a Brilliant Violet 605 were bought from BD Biosciences (San Jose, California, USA). Mouse F4/80 Superbright 702, FoxP3 PE and nestin monoclonal antibodies were purchased from Thermo Fisher. I-BET726 was purchased from Millipore Sigma (Burlington, Massachusetts, USA) and JQ1 was purchased from Tocris (Minneapolis, Minnesota, USA). Live/dead fixable aqua dead cell stain kit and Brilliant stain buffer were purchased from Thermo Fisher.

Wu X., Nelson M., Basu M., Srinivasan P., Lazarski C., Zhang P., Zheng P, & Sandler A.D. (2021). MYC oncogene is associated with suppression of tumor immunity and targeting Myc induces tumor cell immunogenicity for therapeutic whole cell vaccination. Journal for Immunotherapy of Cancer, 9(3), e001388.

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Activation and Expansion of Mouse T-Cells

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Anti (α)-mouse CTLA-4, and mouse IgG2b isotype antibodies were purchased from BioXCell (West Lebanon, NH). COG133 and JQ1 were purchased from Tocris (Minneapolis, MN). Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation kit, Vybrant™ DyeCycle™ Violet Stain kit, SYTOX™ red dead cell stain kit, CellTrace™ far red cell proliferation kit, Live/Dead fixable aqua dead cell stain kit, Brilliant stain buffer and mouse IL-2 Carrier-Free recombinant protein were purchased from Thermo Fisher (Waltham, MA).

Wu X., Srinivasan P., Basu M., Zhang P., Saruwatari M., Thommandru B., Jacobi A., Behlke M, & Sandler A. (2022). Tumor Apolipoprotein E is a key checkpoint blocking anti-tumor immunity in mouse melanoma. Frontiers in Immunology, 13, 991790.

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Cryopreserved PBMC Immunophenotyping

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Cryopreserved peripheral blood mononuclear cell (PBMC) from human, cynomolgus monkey or mouse donors were thawed prior to addition of live/dead viability dye, Fc block, and staining solution. Briefly 0.3–1×10⁶ cells were stained with FVS700 for viability according to manufacturer’s recommendations and blocked with mouse or human Fc block antibody. Next, cells were incubated with CLN-978 conjugated to APC for 60 min at 37°C in the dark and stained with fluorescence-conjugated antibodies. Mouse and non-human primate (NHP) cells were diluted in Brilliant Stain Buffer and FBS Stain Buffer (BD Biosciences), whereas human cells were diluted in Brilliant Stain Buffer and phosphate-buffered saline (Thermo Fisher). After staining, cells were fixed with FluoroFix Buffer (BioLegend) and stored at 2–8°C in the dark until acquisition by flow cytometry. CD4+, CD8+ T cells and CD20+ B cells were identified and gated.

Meetze K., Mehta N.K., Li B., Findlay H.M., London T.B., Patakas A., Michaelson J.S, & Baeuerle P.A. (2023). CLN-978, a novel half-life extended CD19/CD3/HSA-specific T cell-engaging antibody construct with potent activity against B-cell malignancies with low CD19 expression. Journal for Immunotherapy of Cancer, 11(8), e007398.

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