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Fixable viability stain 510

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Fixable Viability Stain 510 is a fluorescent dye that can be used to detect and quantify the percentage of viable cells in a sample. The stain penetrates the cell membrane of dead or dying cells, allowing their identification by flow cytometry or microscopy.

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Lab products found in correlation

Lsrfortessa, by BD (9 mentions) Golgistop, by BD (4 mentions) Facsdiva, by BD (4 mentions) Mcd45 af700, by BD (4 mentions) Hcd3 buv805, by BD (4 mentions) Hcd4 buv395, by BD (4 mentions) Hcd8 percp cy5, by BD (4 mentions) Hcd45 fitc, by BD (4 mentions) Fc block, by BD (4 mentions) Fixation permeabilization buffer, by Thermo Fisher Scientific (3 mentions) Facscanto 2, by BD (3 mentions) Stain buffer, by BD (3 mentions) Fortessa, by BD (3 mentions) F4 80 bv421, by BD (3 mentions) Cd86 pe, by BD (3 mentions) Facsdiva software, by BD (3 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions) Alexa fluor 488 hamster anti mouse cd3e, by BD (2 mentions) Pe cy7 rat anti mouse cd4, by BD (2 mentions) Apc h7 rat anti mouse cd8a, by BD (2 mentions)

94 protocols using fixable viability stain 510

1

CHI3L1 Protein Effects on Cell Viability

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Splenocyte, astrocyte and mixed glial cultures were untreated or treated with vehicle (PBS) or diverse concentrations of mouse recombinant CHI3L1 protein (R&D Systems) for 6, 24 and 48 hours. The effect of CHI3L1 on cell death was assessed using Fixable Viability Stain 510 (BD Biosciences) and flourochrome-labeled anti-CD45 mAb (#561869, BD Biosciences) for splenocytes or Fixable Viability Stain 510 (BD Biosciences) and anti-CD11b mAb (#25-0112-81, eBioscience) for microglia. No lineage-specific marker was used for astrocyte analysis due to the purity of astrocyte culture. Samples were acquired with in a CytoFLEX (Beckman Coulter) flow cytometer and data analysis was performed with CytExpert 2.3 software (CytoFLEX/software">https://www.beckman.es/flow-cytometry/instruments/CytoFLEX/software).
Matute-Blanch C., Calvo-Barreiro L., Carballo-Carbajal I., Gonzalo R., Sanchez A., Vila M., Montalban X, & Comabella M. (2020). Chitinase 3-like 1 is neurotoxic in primary cultured neurons. Scientific Reports, 10, 7118.
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2

Maternal Immune Phenotyping During Pregnancy

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Maternal peripheral blood (150 μL) and leukocyte suspensions from the myometrium were stained using the LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Life Technologies) or Fixable Viability Stain 510 (BD Biosciences) prior to incubation with extracellular and intracellular mAbs, as described above. Immunophenotyping included the identification of surface markers (MHC-II, CD80, CD86, CD206, and CD62L) and cytokines (IFNγ, TNFα, IL-10, and TGFβ) expressed by viable CD11b+Ly6G+OVA- or CD11b+Ly6G+OVA+ cells and CD11b+F4/80+OVA- or CD11b+F4/80+OVA+ cells in the maternal peripheral blood at mid (10.5 dpc) and late (16.5 dpc) pregnancy. The expression of the same cell surface markers and cytokines were evaluated on viable CD11b+Ly6G+OVA+ cells and CD11b+F4/80+OVA+ cells in the myometrial tissues at mid (10.5 dpc) and late (16.5 dpc) pregnancy. The expression of the MHC class I molecules H2Kb and H2Kd was evaluated on viable CD11b+Ly6G+OVA+ and CD11b+F4/80+OVA+ cells from the maternal peripheral blood (50 μL) at mid-gestation (10.5 dpc). Data were analyzed using FlowJo software v10.
Arenas-Hernandez M., Romero R., Gershater M., Tao L., Xu Y., Garcia-Flores V., Pusod E., Miller D., Galaz J., Motomura K., Schwenkel G., Para R, & Gomez-Lopez N. (2021). Specific Innate Immune Cells Uptake Fetal Antigen and Display Homeostatic Phenotypes in the Maternal Circulation. Journal of leukocyte biology, 111(3), 519-538.
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3

Multicolor Flow Cytometry Analysis of Immune Markers

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PBMCs (1x106 cells in each tube) were stained with Alexa Fluor 488 hamster anti-mouse CD3e (BD Biosciences, clone 145-2C11), Pe-Cy7 rat anti-mouse CD4 (BD Biosciences, clone GK1.5), APC-H7 rat anti-mouse CD8a (BD Biosciences, clone 53–6.7), APC hamster anti-mouse PD-1 (BD Biosciences, clone J43), and PE hamster anti-mouse CTLA (BD Biosciences, clone UC10-4F10-11), Fixable Viability Stain 510 (BD Biosciences, cloneR35-95). Corresponding isotype control for each antibody was prepared for appropriate setting of gates during multicolor flow cytometry analysis. All antibodies were pre-titrated for optimal working concentration. Data were acquired on an 8-color FACSCanto II immunocytometry system (BD Biosciences) with BD FACSDiva software (BD Bioscience). Data were exported from BD FACSDiva and analyzed using Flowjo software version 10 (Tree Star, Oregon, USA).
See J.X., Chandramathi S., Abdulla M.A., Vadivelu J, & Shankar E.M. (2017). Persistent infection due to a small-colony variant of Burkholderia pseudomallei leads to PD-1 upregulation on circulating immune cells and mononuclear infiltration in viscera of experimental BALB/c mice. PLoS Neglected Tropical Diseases, 11(8), e0005702.
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4

Multiparametric Flow Cytometry of Immune Cells

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For flow cytometry staining, dissociated cells were incubated with a fixable viability stain 510 (BD Biosciences) followed by staining with primary antibodies directed against CD45 (30-F11), CD11b (M1/70), Gr1 (RB6–8C5), F4/80 (BM8), CD206 (C068C2), MHCII (AF6–120.1), CD44 (IM7), PDL1 (MIH5), TIGIT (1G9), and CD69 (H1.2F3) obtained from BD Biosciences; CD3 (145–2C11), CD4 (GK1.5), CD8 (53–6.7), and pSykY348 (moch1ct) from eBioscience, PD1 (29F.1A12), and CD62 L (MEL-14) from BioLegend. Samples were acquired on BD Fortessa or LSRII and data were analyzed using FlowJo software version 10 (TreeStar).
Rohila D., Park I.H., Pham T.V., Weitz J., Hurtado de Mendoza T., Madheswaran S., Ishfaq M., Beaman C., Tapia E., Sun S., Patel J., Tamayo P., Lowy A.M, & Joshi S. (2023). Syk Inhibition Reprograms Tumor-Associated Macrophages and Overcomes Gemcitabine-Induced Immunosuppression in Pancreatic Ductal Adenocarcinoma. Cancer Research, 83(16), 2675-2689.
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5

Characterizing Regulatory T Cell Populations

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Flow cytometry analyses were performed as described previously (Jang et al., 2019 (link); Kim et al., 2019 (link)). Briefly, MLN tissues were smashed and filtered using a cell strainer (100 μm pore size (SPL Life Sciences Co., Ltd., Pocheon-si, Gyeonggi-do, Republic of Korea) and isolated T cells were subjected to a FC gamma receptor blocking and surface staining for 30 min at 4°C. To identify the live cells, T cells were stained with Fixable Viability Stain 510 (FVS510; BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Moreover, T cell surface were stained with CD3+ fluorescein isothiocyanate (145-2C11; BD Biosciences), CD4+ Percep-Cyanine 5.5 (RM4-5; BD Biosciences) and CD25+ phycoerythrin (PC61; BD Biosciences) following the manufacturer's instructions. T cells were permeabilized with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for intracellular staining and were stained with Foxp3+ Alexa Flour 647 (MF23; BD Biosciences) following the manufacturer's instructions. IgG isotypes (BD Biosciences) were used as a control. The CD4+CD25+Foxp3+ T cell population was counted using a BD FACSVerse™ Flow Cytometer (BD Biosciences).
Kim W.K., Jang Y.J., Han D.H., Seo B., Park S., Lee C.H, & Ko G. (2019). Administration of Lactobacillus fermentum KBL375 Causes Taxonomic and Functional Changes in Gut Microbiota Leading to Improvement of Atopic Dermatitis. Frontiers in Molecular Biosciences, 6, 92.
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6

CD44v6 Expression in AML Cells

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When the target cells were cell lines or T cells, incubation of the cells for 30 min was done with CD44v6‐PE (BD Bioscience, USA) or isotype control mouse IgG1‐PE (BD Bioscience, USA). When the target cells were peripheral blood mononuclear cells (PBMCs) from AML patients or healthy donors, the PBMCs were incubated with the following antibodies (BD Bioscience, USA): Fixable Viability Stain 510, CD117‐PE‐CY7, CD34‐APC, CD45‐APC‐CY7, HLA‐DR‐BV421, CD44v6‐PE or mouse IgG1‐PE. The samples were all detected by BD LSRFortessa X‐20 flow cytometry and analysed by FlowJo software.
Tang L., Huang H., Tang Y., Li Q., Wang J., Li D., Zhong Z., Zou P., You Y., Cao Y., Kong Y., Guo A., Zhou S., Li H., Meng F., Xiao Y, & Zhu X. (2022). CD44v6 chimeric antigen receptor T cell specificity towards AML with FLT3 or DNMT3A mutations. Clinical and Translational Medicine, 12(9), e1043.
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7

Multi-Omic Analysis of Immune Cells

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Peripheral blood was sampled in heparin and red blood cells were lysed.
Samples were stained with anti-CD45.2, CD3, Ly6C, Ly6G and CD11b antibodies. On
the CD45+ CD3- population, neutrophils were defined as Ly6G+CD11b+ and
inflammatory monocytes as Ly6Chi CD11b+. Small intestinal and colonic lamina
propria (LP) lymphocytes were isolated incubating intestinal chunks PBS 5% FCS
1.5 mM EDTA 1mM DTT at 37°C for 15 min to remove epithelial cells. LP
cells were mechanically isolated in RPMI 5% FCS with GentleMACS dissociator. The
cells were permeabilized with FoxP3 intracellular staining kit (eBioscience) and
stained with anti-CD45.2, CD3, CD4, CD25, FoxP3 and Helios antibodies. For Th1
and Th17 detection LP cells were incubated for 4h with PMA (50 ng/ml, Sigma
Aldrich), ionomycin (500 ng/ml, Sigma Aldrich) and GolgiStop (BD Biosciences).
Cells were then stained with anti-CD45.2, CD3, CD4, IL17 PE and IFNγ
antibodies. For mononuclear phagocytes identification LP cells were stained with
anti-CD45.2, CD11, F4/80, CD11c, Ly6G, and Ly6C antibodies. Dead cells were
excluded with the Fixable Viability Stain510 (BD Biosciences). Samples were
acquired at FACSCantoII and Fortessa (BD Biosciences) and analyzed with FlowJo
(Treestar). See Supplementary
Information Table 3 for detailed informations.
Zagato E., Pozzi C., Bertocchi A., Schioppa T., Saccheri F., Guglietta S., Fosso B., Melocchi L., Nizzoli G., Troisi J., Marzano M., Oresta B., Spadoni I., Atarashi K., Carloni S., Arioli S., Fornasa G., Asnicar F., Segata N., Guglielmetti S., Honda K., Pesole G., Vermi W., Penna G, & Rescigno M. (2020). Endogenous murine microbiota member Faecalibaculum rodentium and its human homolog protect from intestinal tumor growth. Nature microbiology, 5(3), 511-524.
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8

Macrophage Isolation and Characterization

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Macrophages were analyzed and sorted using a fluorescence-activated cell sorter (FACS) (BD Biosciences, San Jose, CA, USA) as previously described. Briefly, the AD tissues were dissected, carefully cut into small pieces, and enzymatically digested with collagenase II (1.5 mg/ml), elastase (0.25 mg/ml), and DNase I (0.5 mg/ml) for 1 h at 37°C. After digestion, the tissues were passed through 70-μm cell strainers. After washing, anti-CD16/32 antibody was used to block the non-specific binding. Fixable viability stain 510 (564406, BD Biosciences, San Jose, CA, USA) and the following antibodies were used for flow cytometry: CD45-APC-Cy7 (557659, BD Biosciences), CD11b-PE-Cy7 (552850, BD Biosciences), F4/80-BV421 (565411, BD Biosciences), CD86-PE (553692, BD Biosciences), and CD206-APC (565250, BD Biosciences). For flow cytometric sorting, cells were resuspended in the FACS buffer at 20 × 106 cells/ ml and separated on a MoFlo High-Performance Cell Sorter (Dako Cytomation, Carpinteria, CA, USA). The results were expressed as the absolute number of cells per mg of tissue. Data were analyzed with the FlowJo software (FlowJo LLC, Ashland, OR, USA).
Wang Q., Chen Z., Peng X., Zheng Z., Le A., Guo J., Ma L., Shi H., Yao K., Zhang S., Zheng Z, & Zhu J. (2021). Neuraminidase 1 Exacerbating Aortic Dissection by Governing a Pro-Inflammatory Program in Macrophages. Frontiers in Cardiovascular Medicine, 8, 788645.
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9

Myocardial Inflammatory Cell Profiling via Flow Cytometry

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Single-cell suspensions of CD45-positive inflammatory cells extracted from myocardial tissue were prepared as described above.
To block non-specific binding of the antibody to the Fcγ receptor, the single-cell suspension was first incubated at 4°C with an anti-CD32 antibody (1:400 550273; BD Biosciences, Brøndby, Denmark) for 15 min. Subsequently, the cells were incubated at 4°C with Fixable Viability Stain 510 (1:400 564406; BD Biosciences, Brøndby, Denmark) for 30 min. Next, the cells were incubated with a variety of flow cytometry antibodies (shown in Table 2) at 4°C in the dark for 30 min and stained for cell identification markers. Flow cytometry analysis and cell sorting were performed on a DxP AthenaTM instrument (Cytekbio, Landing Parkway Fremont, USA), and the data were analysed using FlowJo software (Tree Star).

Flow Cytometry Antibody

GeneNo.ProductOrigin
CD68MA5-28262CD68 Monoclonal Antibody (ED1), FITCThermo
CD86551396Rat CD86 PE 24F 100ugBD Pharmingen
CD45561586Rat CD45 APC-Cy7 OX-1 50ugBD Pharmingen
CD11B562102Rat CD11B APC WT.5 50ugBD Pharmingen
Granulocyte13-0570-82Granulocyte Marker Monoclonal Antibody (HIS48), BiotineBioscience
Guo L., Qin G., Cao Y., Yang Y., Dai S., Wang L, & Wang E. (2021). Regulation of the Immune Microenvironment by an NLRP3 Inhibitor Contributes to Attenuation of Acute Right Ventricular Failure in Rats with Pulmonary Arterial Hypertension. Journal of Inflammation Research, 14, 5699-5711.
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10

Comprehensive Single-cell Immunophenotyping

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Single-cell suspension prepared from tumor tissue were pre-incubated with anti-CD16/32 monoclonal antibody (FcR-blocking, BD Biosciences, San Jose, California, USA, clone 2.4G2) and then stained with antibodies against membrane markers for 45 min at 4℃. Dead cells were marked using Fixable Viability Stain 510 (BD Pharmingen, San Diego, California, USA, No. 564406). For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, California, USA, No. 00-5523-00) or the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, No. 88-8824-00) following the manufacture’s instruction. Antibodies were added and incubated for 1 hour at 4℃. Intracellular cytokine staining was performed 4–6 hours after ex vivo stimulation with leucocyte activation cocktail in the presence of GolgiStop (BD Pharmingen, No. 550583) at 37℃. All the fluorescently labeled antibodies used for staining are listed in online supplemental table S1. Data were collected with a BD LSRFortessa and analyzed using FlowJo (V.10.0). According to the isotype and fluorescence-minus-one, the gating strategies for flow cytometry were showed in online supplemental figure S4.
Sun P., Zhang X., Wang R.J., Ma Q.Y., Xu L., Wang Y., Liao H.P., Wang H.L., Hu L.D., Kong X., Ding J, & Meng L.H. (2021). PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8+T cells and promoting fatty acid metabolism. Journal for Immunotherapy of Cancer, 9(8), e003093.
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