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Fixable viability dye efluor 780

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Fixable Viability Dye eFluor 780 is a fluorescent dye used to identify dead cells in flow cytometry and fluorescence microscopy applications. It provides a reliable method for detecting nonviable cells in a sample.

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Cd8 fitc, by BD (1 mentions) Cd86 pe cy7, by BD (1 mentions) Cd3 pe cy7, by BD (1 mentions) Cd45 apc, by BD (1 mentions) Cd25 apc, by BD (1 mentions) Cd86 apc, by BD (1 mentions) Cd11c pe cy7, by BD (1 mentions) B220 biotin, by BD (1 mentions) Biotin cd4, by BD (1 mentions) Foxp3 pe, by BD (1 mentions) Cd8α fitc, by BD (1 mentions) Cd40 pe cy7, by BD (1 mentions) Cd24 pe cy7, by BD (1 mentions) Biotin cd25, by BD (1 mentions) Propidium iodide solution, by BD (1 mentions) Alexa fluor 647 conjugated ova, by Thermo Fisher Scientific (1 mentions) Apc tcr β, by BD (1 mentions) Fitc sirpα, by BD (1 mentions) Brilliant violet 605 cd11c, by BD (1 mentions) Pacific blue ia ie, by BD (1 mentions)

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EFluor 780 (13 citations) Fixable viability dye (9 citations) Viability dye (2 citations)

7 protocols using fixable viability dye efluor 780

1

NY-ESO-1-specific T Cell Activation in Alginate Cryogels

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CD8+ T cells from the
same HLA-A2.1+ donor as the moDCs were transfected, as
previously described27 (link) with mRNA (5 mg/mL,
BioNTech RNA Pharmaceuticals) encoding for a mouse T cell receptor
(specific for the HLA-A2.1-specific NY-ESO-1 epitope SLLWITQC). Transfection
efficiency was determined 1 day after transfection using anti-mouse
TCR-β-FITC (BioLegend) and was typically between 80 and 90%
(Figure S9A). These NY-ESO-1 specific T
cells were stained with CellTrace Violet and were added to activated
moDCs (ratio DCs/T cells as 1:2.5) in alginate cryogels. The supernatant
was taken after 24 h of T cell activation to determine IFNγ
production. After 72 h of activation, cells were collected and stained
with Fixable Viability Dye eFluor 780, CD8-FITC (BD BioScience), and
CD25-PE (BD BioScience).
Weiden J., Schluck M., Ioannidis M., van Dinther E.A., Rezaeeyazdi M., Omar F., Steuten J., Voerman D., Tel J., Diken M., Bencherif S.A., Figdor C.G, & Verdoes M. (2021). Robust Antigen-Specific T Cell Activation within Injectable 3D Synthetic Nanovaccine Depots. ACS Biomaterials Science & Engineering, 7(12), 5622-5632.
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2

Multiparameter Immune Cell Analysis

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The following antibodies were purchased from BioLegend, eBioscience, or BD Biosciences, and used for flow cytometry or enrichment of specific cell types: FITC-Sirpα, PE/Cy7-Sirpα PerCP/Cy5.5-B220, PE/Cy7-CD11c, Brilliant Violet 605-CD11c, Pacific Blue-IA/IE, FITC-YAe, PerCP/Cy5.5-CD45.1, APC-CD45.2, PE/Cy7-CD86, APC-CD86, PE-PD-L1, APC-CD200, PE/Cy7-CD40, PE-Foxp3, Pacific Blue-Foxp3, PerCP/Cy5.5-Thy1.1, APC-TCRβ, FITC-CD8α, Alexa700-CD8α, Alexa700-CD4, Brilliant Violet 605-CD4, Brilliant Violet 605-CD25, APC-CD25, PE/Cy7-CD3, PE/Cy7-CD24, Propidium iodide solution, Fixable Viability Dye eFluor780, Biotin-CD8β, Biotin-CD4, Biotin-CD25, and Biotin-B220. AlexaFluor647-conjugated OVA and crimson bead (0.02 μm) were purchased from ThermoFisher Scientific. Streptavidin-RapidSpheres isolation kit and CD11c-MicroBeads were purchased from STEMCELL technologies and Miltenyi Biotec, respectively.
Oh J., Wu N., Barczak A.J., Barbeau R., Erle D.J, & Shin J.S. (2018). CD40 Mediates Maturation of Thymic Dendritic Cells Driven by Self-reactive CD4+ Thymocytes and Supports Development of Natural Regulatory T Cells. Journal of immunology (Baltimore, Md. : 1950), 200(4), 1399-1412.
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3

Sorting of Naïve and Memory B Cells

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B cells were incubated with an antibody cocktail containing L/D marker (eBioscience Fixable Viability Dye eFluor 780) and antibodies CD10 BUV737 (BD #612826), CD20 PECy7 (BioLegend #302312) and CD27 BV711 (BioLegend #302834) and IgD BUV395 (BD #563813) for 30 min at 4°C. Labelled B cells were sorted into naïve (CD10−CD20+CD27−IgD+) and memory (CD10−CD20+CD27+ and CD10−CD20+CD27−IgD−) B cells using the FACSAria™ Fusion sorter (BD Biosciences) (gating strategy shown in Figure S1A).
Lee J.L., Fra‐Bido S.C., Burton A.R., Innocentin S., Hill D.L, & Linterman M.A. (2022). B cell‐intrinsic changes with age do not impact antibody‐secreting cell formation but delay B cell participation in the germinal centre reaction. Aging Cell, 21(9), e13692.
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4

CD8+ T Cell Isolation from Tumor

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CD8+ T cells were isolated from tumor single-cell suspensions using flow sorting (FACS Aria cell sorter, BD Biosciences). Single-cell suspension from tumor tissues was incubated with PBS containing Mouse BD Fc Block, eBioscience Fixable Viability Dye eFluor 780 and anti-mouse CD8a Pacific Blue (558106, Clone: 53-6.7, Rat IgG2a, κ, BD Biosciences). CD8+ T cells were sorted with the purity >90%.
Martins Nascentes Melo L., Herrera-Rios D., Hinze D., Löffek S., Oezel I., Turiello R., Klein J., Leonardelli S., Westedt I.V., Al-Matary Y., Egea-Rodriguez S., Brenzel A., Bau M., Sucker A., Hadaschik E., Wirsdörfer F., Hanenberg H., Uhlenbrock N., Rauh D., Poźniak J., Rambow F., Marine J.C., Effern M., Glodde N., Schadendorf D., Jablonska J., Hölzel M, & Helfrich I. (2023). Glucocorticoid activation by HSD11B1 limits T cell-driven interferon signaling and response to PD-1 blockade in melanoma. Journal for Immunotherapy of Cancer, 11(4), e004150.
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5

Comprehensive Immunophenotyping of Immune Cells

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Fluorochrome-conjugated monoclonal/polyclonal antibodies used in our studies were anti-mouse CD4 (GK1.5, BD Biosciences), anti-mouse CD25 (PC61.5l, eBioscience), anti-mouse Foxp3 (FJK-16s, eBioscience), anti-mouse CD8 (53-6.7, eBioscience), anti-human CD19 (H1B19, eBioscience), anti-human EGFR (AY13, BioLegend), anti-mouse CD45.1 (A20, eBioscience), anti-mouse CTLA-4 (UC10-4B9, eBioscience), anti-mouse neuropilin 1 (eDS304M, eBioscience), anti-mouse Lag-3 (C9B7W, eBioscience), anti-mouse CD11c (N418, eBioscience), anti-mouse IFN-γ (XMG1.2, eBioscience), anti-mouse TNF-α (MP6-XT22, eBioscience), anti-mouse Fas (SA367H8, BioLegend), anti-mouse FasL (MFL3, eBioscience), anti-mouse perforin (eBioMAK-D, eBioscience), anti-mouse GzB (NGZB, eBioscience), anti-mouse GzA (3G8.5, BioLegend), anti-mouse CD107α (1D4B, eBioscience), anti-mouse CD71 (R17217, eBioscience), anti-mouse CPT1a (8F6AE9, Abcam), anti-mouse Glut1 (ER3915, Abcam), and Fixable viability dye eFluor 780 (BD Biosciences). Intracellular staining was performed using the fixation/permeabilization concentrate (catalog 5123-43) and diluent (catalog 5223-56) buffer solutions and IC buffer (catalog 8333-56), according to the eBioscience Foxp3 staining kit. Stained cells were analyzed on an LSR Fortessa flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).
Bolivar-Wagers S., Loschi M.L., Jin S., Thangavelu G., Larson J.H., McDonald-Hyman C.S., Aguilar E.G., Saha A., Koehn B.H., Hefazi M., Osborn M.J., Jensen M.C., Wagner J.E., Pennell C.A, & Blazar B.R. (2022). Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses. JCI Insight, 7(17), e160674.
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6

CAR-NK and CAR-T Cell Surface Phenotyping

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Cell viability was determined using Fixable Viability Dye eFluor 780 or Fixable Viability Stain 510 (BD Biosciences) according to the manufacturer's instructions. Cells were then stained with anti-human fluorescently labeled extracellular antibodies for the indicated extracellular markers in FACS buffer (0.2% BSA in PBS) at 4°C for 30 min. Detection of the HER2 CAR construct on the surface of human expanded CAR-NK cells, CAR-NK-92 cells, and CAR-T cells was determined by incubation with 2.5 μg of HER2 Fc chimeric protein (R&D Systems) in FACS buffer for 30 min at room temperature, followed by staining with a PE- or FITC-conjugated anti-human IgG Fc antibody (BioLegend). Transduced cells were also stained with APC-conjugated anti-NGFR (CD271) antibody (BioLegend) or VioBrightFITC-conjugated anti-NGFR antibody. All stained samples were fixed for 1 hr with 1% paraformaldehyde. All flow cytometry was conducted on a BD LSRFortessa or BDLSRII cytometer (BD Biosciences) and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR).
Portillo A.L., Hogg R., Poznanski S.M., Rojas E.A., Cashell N.J., Hammill J.A., Chew M.V., Shenouda M.M., Ritchie T.M., Cao Q.T., Hirota J.A., Dhesy-Thind S., Bramson J.L, & Ashkar A.A. (2021). Expanded human NK cells armed with CAR uncouple potent anti-tumor activity from off-tumor toxicity against solid tumors. iScience, 24(6), 102619.
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7

T Cell Activation and Proliferation Assay

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On day 1, T cells were transferred to v-bottom 96-well plates (Corning)
and stained with the Zombie Violet Fixable Viability Kit (Biolegend).
To determine T cell activation, we performed cell surface staining
with CD8-BV510, CD4-APC-Cy7, CD69-PE, and CD25-PE-Cy7 (all BD Biosciences).
On day 3, we determined T cell proliferation by transferring T cells
to v-bottom 96-well plates and staining with Fixable Viability Dye
eFluor 780 (BD) followed by cell surface staining with CD4-PE (BD
Biosciences) and CD8-BV510. All samples were acquired on the FACS
Verse (BD Biosciences). The mean cell cycle of all T cells was determined
as a measure for the average number of cell proliferation cycles.
The mean cycle was calculated with the formula log2(f), where f is the CellTrace Violet mean fluorescence
intensity (MFI) of all nonproliferated T cells divided by the CellTrace
Violet MFI of all T cells.
Hammink R., Weiden J., Voerman D., Popelier C., Eggermont L.J., Schluck M., Figdor C.G, & Verdoes M. (2021). Semiflexible Immunobrushes Induce Enhanced T Cell Activation and Expansion. ACS Applied Materials & Interfaces, 13(14), 16007-16018.
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