Fixable viability kit

Manufactured by BioLegend

Sourced in United States

The Fixable Viability Kit is a lab equipment product that allows for the identification of live and dead cells in a sample. It provides a straightforward method for distinguishing between viable and non-viable cells using flow cytometry.

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10 protocols using fixable viability kit

Multiparametric Flow Cytometry Analysis

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Single cell suspensions were stained with different combinations of fluorochrome-conjugated antibodies (Table S1 in Supplementary Material), and dead cells were excluded using Fixable Dead Cell Stain Kit (Thermo Fisher) or Fixable Viability Kit (BioLegend). For intracellular staining of IFN-γ and TNF, cells were surface stained and fixated with 2% PFA (EMS Sciences), followed by treatment with Permeabilizing Solution 2 (BD Biosciences). Data were acquired on an LSR Fortessa (BD Biosciences) and analyzed with FlowJo vX (FlowJo LLC) software. ARIA or ARIA II instruments (both BD Biosciences) were used for cell sorting experiments.

Hammer Q., Rückert T., Dunst J, & Romagnani C. (2018). Adaptive Natural Killer Cells Integrate Interleukin-18 during Target-Cell Encounter. Frontiers in Immunology, 8, 1976.

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Flow Cytometric Analysis of MSC and T-Cell Markers

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The stem cell surface markers of the MSCs were administered using flow cytometry, which was stained with a Fixable Viability Kit (Biolegend, San Diego, CA) and Mouse MSC Analysis Kit (Cyagen, Santa Clara, CA) for 30 min according to the instructions. The CFSE-labeled MSCs were harvested on the 2nd three-day cultivation and detected by flow cytometry, with additional staining using the Fixable Viability Kit. The spleen and kidney single-cell suspensions from experimental mice were first stained via fluorophore-conjugated antibodies, including BV510 Fixable Viability Kit (Biolegend, San Diego, CA), FITC anti-CD45 (Biolegend, San Diego, CA), PerCP/Cy5.5 anti-CD4 (Biolegend, San Diego, CA), APC anti-CD25 (Biolegend, San Diego, CA) for 30 min at 4 °C, follow by PE anti-Foxp3 (Biolegend, San Diego, CA), and PE/Cy7 anti-mTOR (Invitrogen, Carlsbad, CA) for 2 h at 4 °C. The CD4⁺T cells cultured in vitro were stained via BV510 Fixable Viability Kit, PerCP/Cy5.5 anti-CD4, and APC anti-CD25 antibodies for 30 min at 4 °C. The CD4⁺CD25⁺CD127^–Tregs sorted by fluorescence activated cell sorting (FACS) were stained via BV510 Fixable Viability Kit, PerCP/Cy5.5 anti-CD4, APC anti-CD25, and PE anti-CD127 (Biolegend, San Diego, CA) antibodies for 30 min at 4 °C.

Luo Y., Zhang K., Wu J., Zeng H., Chen J., Zhang P., Guo J., Xu C., Niu X., Cheuk Y.C., Xu S., Cao Y., Zhao Y., Zhu D., Wang X, & Rong R. (2023). Periosteum-derived mesenchymal stem cell alleviates renal fibrosis through mTOR-mediated Treg differentiation. Renal Failure, 45(1), 2212079.

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Single-Cell Analysis of Immune Cell Profiles in Oral Squamous Cell Carcinoma

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The OSCC tissues were cut and digested into single cells. Dissociated cells (from the OSCC tissues or in vitro experiment) were filtered with a 70-μm cell strainer. After being stained with the Fixable Viability kit (BioLegend catalog no. L423105), single cell suspensions were blocked with anti-CD16/32 antibody (BioLegend catalog no. 101320) and incubated with the following antibodies: CD45 (BioLegend catalog no. 103140), CD3 (BioLegend catalog no. 100204), TCR γδ (BioLegend catalog no. 118118; BioLegend catalog no. 107508), PD-1 (BioLegend catalog no. 135206; BioLegend catalog no. 114101), IL-17A (BioLegend catalog no. 506916), CD11b (BioLegend catalog no. 101228), F4/80 (BioLegend catalog no. 123130), CD206 (BioLegend catalog no. 141706). For intracellular cytokine staining, single cell suspensions were stimulated with cell activation cocktail (BioLegend catalog no. 423303) at 37°C in a 5% CO₂ incubator for 6 h before surface staining, fixation (BioLegend catalog no. 420801), and permeabilization (BioLegend catalog no. 421002). Flow cytometry was performed on an Attune NxT flow cytometer (Invitrogen Attune NxT flow cytometry software), and data were analyzed by the FlowJo (V10.8) software.

Wei W., Li J., Shen X., Lyu J., Yan C., Tang B., Ma W., Xie H., Zhao L., Cheng L., Deng Y, & Li Y. (2022). Oral Microbiota from Periodontitis Promote Oral Squamous Cell Carcinoma Development via γδ T Cell Activation. mSystems, 7(5), e00469-22.

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Activation and Analysis of Human T Cells

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Human CD3⁺ T cells were activated by CD3/CD28 T Cell Activator (StemCell) for 48 h in the presence of 10 μg/mL CD25 antibody in 1640 + 10% FBS (Gibco) medium. After staining by Fixable Viability Kit (Biolegend, 423105) for 15 min, cells were washed by two times. Cells were then stained by mixture of anti-human CD3, CD4 and CD8 fluorescent-labeled antibody at 4 °C for 20 min and washed. After resuspending by 1 × Fixation/Permeabilization buffer, cells were incubated in dark for 30 min at room temperature and then resuspended by 1 × Permeabilization buffer. After centrifugation, cells were stained by Ki67 and Granzyme B fluorescent-labeled antibody for 30 min and then analyzed by Beckman CytoFLEX flow cytometry. Experiments were performed in triplicate, value = mean ± SEM.

Song D., Liu X., Dong C., Wang Q., Sha C., Liu C., Ning Z., Han J., Liu H., Zong M., Zhao Y., Li Y., Liu G., Shao X, & Dou C. (2021). Two novel human anti-CD25 antibodies with antitumor activity inversely related to their affinity and in vitro activity. Scientific Reports, 11, 22966.

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Cell Identification and Sorting for Transcriptomics

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Cell populations were identified using the antibodies listed in the Key Resources Table above, after gating for singlets and live cells. Cells were sorted for transcriptomic analysis using the BD-FACS ARIA, with marker expression measured on the BD-LSRII. For Sall1^gfp/+ cell sorting, flow cytometry was performed using an LSRII Fortessa. Dead cells were excluded with the Fixable Viability Kit (Biolegend) or using Hoechst staining. Data were analyzed using FlowJo software (Treestar).

Thion M.S., Low D., Silvin A., Chen J., Grisel P., Schulte-Schrepping J., Blecher R., Ulas T., Squarzoni P., Hoeffel G., Coulpier F., Siopi E., David F.S., Scholz C., Shihui F., Lum J., Amoyo A.A., Larbi A., Poidinger M., Buttgereit A., Lledo P.M., Greter M., Chan J.K., Amit I., Beyer M., Schultze J.L., Schlitzer A., Pettersson S., Ginhoux F, & Garel S. (2018). Microbiome Influences Prenatal and Adult Microglia in a Sex-Specific Manner. Cell, 172(3), 500-516.e16.

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Comprehensive Murine Immune Cell Analysis

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Composition of murine immune cells was analyzed using the following antibodies: CD3 (145-2C11; BioLegend), CD19 (6D5; BioLegend), CD20 (SA275A11; BioLegend), CD11b (M1/70; BioLegend), CD11c (N418; BioLegend), CD45 (30-F11; BioLegend), Ly6C (HK1.4; BioLegend) and Ly6G (1A8: BioLegend). B cell maturation was analyzed using the following antibodies: CD19 (6D5; BioLegend), CD21 (7G6; BD Bioscience), CD23 (B3B4; BD Bioscience), CD93 (AA4.1; BioLegend), CD45R/B220 (RA3-6B2; BioLegend), IgD (11-26c.2a; BioLegend) and IgM (AF6-78; BD Bioscience). Monocyte, macrophage and microglia activation, differentiation and molecules involved in antigen presentation were determined using: CD40 (3/23; BD Bioscience), CD68 (FA-11; BioLegend), CD69 (H1.2F3; BioLegend), CD80 (16-10A1; BioLegend), CD86 (GL-1; BioLegend), MHCII (AF6-120.1; BioLegend) and PD-L1 (MIH5; eBioscience). Fc receptors were blocked using monoclonal antibody specific for CD16/ CD32 (93; BioLegend). Dead cells were stained with a fixable viability kit (BioLegend). Samples were acquired on a BD LSR Fortessa (BD Bioscience). All data evaluation was performed using FlowJo software (FlowJo LLC, Ashland, USA).
Intracellular proteins were analyzed using the BD PhosFlow protocol and analyzed using the following antibodies: BTK (53/BTK; BD Bioscience), pBTK (N35-86, BD Bioscience), iNOS (W16030C, Biolegend) Arg1 (A1exF5, eBioscience).

Geladaris A., Torke S., Saberi D., Alankus Y.B., Streit F., Zechel S., Stadelmann-Nessler C., Fischer A., Boschert U., Häusler D, & Weber M.S. (2024). BTK inhibition limits microglia-perpetuated CNS inflammation and promotes myelin repair. Acta Neuropathologica, 147(1), 75.

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Isolation and Analysis of Mouse Regulatory T Cells

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Cells from the MLNs were extracted as previously mentioned.^{65 (link)} The mice’s MLNs were gently broken up
and run through a cell strainer with a 40 micron pore size. The cells
were obtained and resuspended in cell staining buffer (Elabscience,
China) for further staining. A Fixable Viability Kit (423102; BioLegend,
USA) was used to stain live cells, and cell surfaces were stained
with fluorescein isothiocyanate anti-mouse CD4 antibody (E-AB-F1097C;
Elabscience, China) and allophycocyanin anti-mouse CD25 antibody (E-AB-F1102E;
Elabscience, China). Fixation/permeabilization buffer (Thermo Scientific,
USA) was used to permeabilize the cells, and the intracellular Foxp3
was stained using the phycoerythrin anti-mouse Foxp3 antibody (E-AB-F1238D;
Elabscience, China). According to the manufacturer’s instructions,
the fluorescent antibodies were applied. A FACS Arial II flow cytometer
(BD Biosciences, USA) was used to detect cells, and FlowJo software
was used to conduct the analysis.

Liao Y., Wu X., Luo W., Chen J., Huang Y., Ma K., Zhang C., Wang J., Yang Y., Deng M, & Wang X. (2023). Azelaic Acid Regulates the Renin–Angiotensin System and Improves Colitis Based on Network Pharmacology and Experimentation. ACS Omega, 8(17), 15217-15228.

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Epigenetic Modulation Enhances CAR-T Cytotoxicity

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A 10:1 ratio of effector cells: target cells (E: T) was used in co-culturing NT or CAR-T cells pretreated with Dec (0.05, 0.1 μM) and Aza (0.5, 1 μM) for 6 days with CFSE-labeled (BD Biosciences, Cat#565082) CD44v6⁺ MV4-11 cells for 24 h. The same E: T ratio and co-culture time were used in incubating Dec 1 μM or Aza 1 μM pretreated AML cells with CD44v6 CAR-T cells. Dead cells were detected via flow cytometry using Fixable Viability Kit (Biolegend, Cat#423102) staining. The percentage of dead CFSE-positive cells was used to evaluate the cytotoxicity.

Tang L., Kong Y., Wang H., Zou P., Sun T., Liu Y., Zhang J., Jin N., Mao H., Zhu X., Wang J., Meng F, & You Y. (2023). Demethylating therapy increases cytotoxicity of CD44v6 CAR-T cells against acute myeloid leukemia. Frontiers in Immunology, 14, 1145441.

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Profiling PCa Cells in Co-culture

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OBs and PCa cells (PC3 and DU145) were removed from co-culture experiments with trypsin and suspended in flow buffer (1X PBS + 2%FBS). Cells were then labeled with a Fixable Viability Kit according to package instructions (Biolegend, Cat# 423111), HLA-A,B,C (Biolegend, Cat# 311426), and in the case of parental PCa cells anti-human Ki67 (Biolegend, Cat# 350514) for 30 min at room temperature. Cells were then washed 2x with flow buffer and re-suspended in flow buffer for flow analysis. Live cells were gated for HLA-A,B,C prior to analysis of Ki67 or FUCCI cell cycle expression.

Decker A.M., Jung Y., Cackowski F.C., Yumoto K., Wang J, & Taichman R.S. (2017). Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow. Molecular cancer research : MCR, 15(12), 1644-1655.

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Cytokine Expression Profiling by Flow Cytometry

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The cells were stimulated with PMA and Ionomycin (Cell Stimulation Cocktail, Invitrogen, Cat. No. 00-4970-03, lot: 2430454) for 4 hours. Cytokine transport was blocked with Brefeldin A (Invitrogen, Cat. No. 00-4506-51). After staining using a Fixable Viability Kit (Biolegend, lot: B333785) and for the CD4 marker (cat:1540-26, lot:F2212-T406C), the cells were fixed and permeabilized (Invitrogen ref: 00-5523-00 , lot: 2333698) followed by staining with fluorochrome-conjugated anti-mouse antibodies against RORγt (clone:B2D, lot: 2304447), IL-17 (clone:eBio17B7, lot: 2142931), IFN-γ (clone: XMG1.2, lot: 2289505), and Foxp3 (clone:FJK-16s, lot: 2199652) at 1:800 dilution. IL-17, FoxP3, RORγt and IFN-γ expression was quantified using an Attune NxT flow cytometer. Data was analyzed using FlowJo software.

McBride D.A., Dorn N.C., Yao M., Johnson W.T., Wang W., Bottini N, & Shah N.J. (2023). Short-chain fatty acid-mediated epigenetic modulation of inflammatory T cells in vitro. Drug delivery and translational research, 13(7).

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