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Efluor 780

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EFluor 780 is a fluorescent dye used for flow cytometry applications. It is designed to provide a bright and stable fluorescent signal for the detection and analysis of cells or particles.

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Lab products found in correlation

Efluor 780, by Thermo Fisher Scientific (5 mentions) Rat anti mouse siglec f alexa fluor 647 clone e50 2440, by BD (2 mentions) Rat anti mouse ly 6g fitc clone 1a8, by BioLegend (2 mentions) Fluorescein isothiocyanate (fitc), by BioLegend (2 mentions) Cd99 fitc, by BioLegend (2 mentions) Ccr10 apc, by BD (2 mentions) Cd3 af700, by BioLegend (2 mentions) Cd4 pe cy7, by BioLegend (2 mentions) Ccr10 apc, by R&D Systems (2 mentions) Cd45ra pe, by BioLegend (2 mentions) Hla dr apc, by BioLegend (2 mentions) Cd25 apc, by BioLegend (2 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions) Cd69 apc, by BioLegend (2 mentions) Cd69 bv421, by BioLegend (2 mentions) Mouse fitc cd4 and efluor 605 cd44, by BD (2 mentions) Brilliant violet 570 cd4, by BioLegend (2 mentions) Alexa fluor 700 cd44, by BioLegend (2 mentions) Fitc klotho, by Bioss Antibodies (2 mentions) Pe cy7 cd99, by Bioss Antibodies (2 mentions)

Spelling variants of this product

Fixable Viability Dye eFluor 780 (7 citations) APC-eFluor 780-labeled anti-mouse Ly-6G (2 citations) CD45RA Alexa eFluor 780 (2 citations) APC-eFluor 780 (2 citations)

13 protocols using efluor 780

1

Flow Cytometric Analysis of BALF

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Differential cell counts in BALF were determined by flow cytometry as described [26 (link), 27 (link)]. Briefly, BALF cells were resuspended in FACS buffer (5% BSA, 0.35 mM EDTA, 0.01% NaN3). Cell staining was performed according to manufacturer’s recommendations using fixable viability dye eFluor 780, rat anti mouse-CD16/CD32 (clone 93), rat anti mouse-CD45 PE-eFluor610 (30-F11), rat anti-mouse CD11b PE-Cy7 (clone M1/70), rat anti-mouse Siglec-F Alexa Fluor 647 (clone E50-2440) (all from BD Biosciences, San Jose, CA); anti-mouse CD64 PerCP-Cy5-5 (clone X54-5/7.1) and rat anti-mouse Ly-6G FITC (clone 1A8) (both from Biolegend, San Diego, CA). Flow cytometry analysis was performed using a FACSCANTO II (Becton Dickinson, Franklin Lakes, NJ) and data were analyzed using FlowJo software (Becton Dickinson). The gating strategy that was used to analyze the different populations is shown in supplementary Figure S1. Fibrotic macrophage population was defined as CD45 + CD64 + SigecFlowCD11bhi; classical alveolar macrophages were defined as CD45 + CD64 + SiglecFhiCD11blow.
Qin W., Spek C.A., Scicluna B.P., van der Poll T, & Duitman J. (2022). Myeloid DNA methyltransferase3b deficiency aggravates pulmonary fibrosis by enhancing profibrotic macrophage activation. Respiratory Research, 23, 162.
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2

Multiparametric Flow Cytometry Immunophenotyping

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Mouse FITC-CD4 and eFluor 605-CD44 [BD-Pharmingen]; KJ1.26 anti-TCR (Caltag Laboratories), Brilliant Violet 570-CD4 and Alexa Fluor 700-CD44 were from [Biolegend], FITC-Klotho,PE-Cy7-CD99, PE-Cy5-VDR, and PE-Cy7-CNR2 were from [Bioss]; APC-CCR10 and APC-ITGA3 were from [R&D Systems]; Fixable viability dye eFluor 780 was from [eBioscience]. Antibodies for human PBMC staining were as follows: from Biolegend, CD99-FITC,CD69-BV421 and CD69-APC , CD45R0-APC and CD45R0-BV421, CD3-AF700, CD4-Pe/Cy7, CD45RA-PE, Itga3 (CD49c)-PE, IL-7R-BV510, CD25-APC, HLA-DR-APC. From BD Biosciences, CCR10-APC and CCR10-PerCP Cy5.5. and viability dye eFluor 780 as above. Relevant mouse anti human isotype antibodies to IgG1k or IgG2ak were utilized for the following fluorophores: BV421, BV510, FITC, PE, and APC for both isotypes (Biolegend).
Song N., Sengupta S., Khoruzhenko S., Welsh R.A., Kim A., Kumar M.R., Sønder S.U., Sidhom J.W., Zhang H., Jie C., Siliciano R.F, & Sadegh-Nasseri S. (2020). Multiple genetic programs contribute to CD4 T cell memory differentiation and longevity by maintaining T cell quiescence. Cellular immunology, 357, 104210.
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3

Quantifying Golgi Retention using Fluorescence

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Inducible stable cell lines expressing GFP-tagged Golgi enzyme chimeric reporters were cultured in six-well plate format in selection medium containing 60 µg/ml cumate for at least a week before analysis. Once cells reached 80–90% confluency, they were washed once with EDTA solution and incubated in accutase for 2 min at 37°C. Cells were resuspended in selection medium and transferred into a deep 96-well plate. Cells were washed once by centrifugation at 300 g for 5 min, followed by resuspension in ice-cold FACS buffer. Cells were transferred to a round-bottomed 96-well plate and incubated on ice in darkness for 30 min with an AF647-conjugated anti-GFP antibody (1:20, BioLegend) and fixable viability dye eFluor 780 (1:1,000, BD Biosciences) diluted in FACS buffer. Cells were subsequently washed, fixed, and analyzed as described for lectin stains. Furthermore, GFP-negative cells were excluded from analysis, and a ratio of the AF647 signal to the GFP signal was used to derive a quantitative parameter for Golgi retention.
Welch L.G., Peak-Chew S.Y., Begum F., Stevens T.J, & Munro S. (2021). GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles. The Journal of Cell Biology, 220(10), e202106115.
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4

Phenotypic Analysis of T-Cell Subsets

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The procedure for lentivirus infection and coculture has been described above. Then, the cells were harvested and stained with phycoerythrin (PE)-CF594-conjugated antihuman CD4, antihuman CD8 PerCP/Cy5.5, and eFluor™ 780 (eBioscience, CA, USA). In mice, single-cell suspensions of spleens were prepared and stained with antimouse CD4 FITC, PE-Cy7-conjugated antimouse Ki67 (eBioscience, CA, USA), eFluor™ 780, antimouse CD3a BUV395, and antimouse CD8a BV510 (BD Pharmingen, USA). Data were collected by BD LSRFortessa™ Flow Cytometer and analyzed by FlowJo software.
Li Z., Wang R., Wang D., Zhang S., Song H., Ding S., Zhu Y., Wen X., Li H., Chen H., Liu S, & Sun L. (2023). Circulating miR-320b Contributes to CD4+ T-Cell Proliferation in Systemic Lupus Erythematosus via MAP3K1. Journal of Immunology Research, 2023, 6696967.
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5

Quantification of Airway Neutrophils by Flow Cytometry

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Neutrophils in BALF were determined by flow cytometry as described [65 (link)]. Briefly, BALF cells were resuspended FACS buffer (5% BSA, 0.35 mM EDTA, 0.01% NaN3) and stained with fixable viability dye eFluor 780, rat anti mouse-CD45 PE-eFluor610 (30-F11), rat anti-mouse CD11b PE-Cy7 (clone M1/70), rat anti-mouse Siglec-F Alexa Fluor 647 (clone E50-2440), rat anti-mouse Ly-6C Alexa Fluor 700 (clone AL-21) (all from BD Biosciences) and rat anti-mouse Ly-6G FITC (clone 1A8; Biolegend, San Diego, CA) before loading on FACS Calibur (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using FlowJo software (Becton Dickinson). Neutrophils were identified as CD45+/Siglec-F-/CD11b+/Ly6C+/Ly6G+ cells. Examples of the gating strategy for BALF neutrophils are depicted in S9 Fig.
Qin W., Brands X., van’t Veer C., F. de Vos A., Sirard J.C., J. T. H. Roelofs J., P. Scicluna B, & van der Poll T. (2021). Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection. PLoS Pathogens, 17(4), e1009491.
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6

Multiparametric Flow Cytometry Immunophenotyping

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Mouse FITC-CD4 and eFluor 605-CD44 [BD-Pharmingen]; KJ1.26 anti-TCR (Caltag Laboratories), Brilliant Violet 570-CD4 and Alexa Fluor 700-CD44 were from [Biolegend], FITC-Klotho,PE-Cy7-CD99, PE-Cy5-VDR, and PE-Cy7-CNR2 were from [Bioss]; APC-CCR10 and APC-ITGA3 were from [R&D Systems]; Fixable viability dye eFluor 780 was from [eBioscience]. Antibodies for human PBMC staining were as follows: from Biolegend, CD99-FITC,CD69-BV421 and CD69-APC , CD45R0-APC and CD45R0-BV421, CD3-AF700, CD4-Pe/Cy7, CD45RA-PE, Itga3 (CD49c)-PE, IL-7R-BV510, CD25-APC, HLA-DR-APC. From BD Biosciences, CCR10-APC and CCR10-PerCP Cy5.5. and viability dye eFluor 780 as above. Relevant mouse anti human isotype antibodies to IgG1k or IgG2ak were utilized for the following fluorophores: BV421, BV510, FITC, PE, and APC for both isotypes (Biolegend).
Song N., Sengupta S., Khoruzhenko S., Welsh R.A., Kim A., Kumar M.R., Sønder S.U., Sidhom J.W., Zhang H., Jie C., Siliciano R.F, & Sadegh-Nasseri S. (2020). Multiple genetic programs contribute to CD4 T cell memory differentiation and longevity by maintaining T cell quiescence. Cellular immunology, 357, 104210.
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7

Immunophenotyping of B cells and DCs

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B lymphocytes were stained with anti-CD38 APC (Clone HIT2, Biolegend) and anti-IgA biotin (Novex). After washing, streptavidin alexa-488 and fixable viability dye eFluor780 (eBioscience) were used. Differentiated monocyte-derived dendritic cells were incubated with an CD103 APC (Clone Ber-ACT8, BD Bioscience) and fixable viability dye eFluor780. Samples were acquired on LSR-Fortessa X20 (BD Bioscience) and data was analyzed with Flowjo software (Tree Star). Data represented in histograms are normalized to mode, meaning data is normalized to the highest value per condition.
Bos A.V., Erkelens M.N., Koenders S.T., van der Stelt M., van Egmond M, & Mebius R.E. (2021). Clickable Vitamins as a New Tool to Track Vitamin A and Retinoic Acid in Immune Cells. Frontiers in Immunology, 12, 671283.
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8

Multiparametric Flow Cytometry Analysis

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Peripheral blood was collected from mice by puncture of the retro-orbital venous plexus directly into flow tubes containing FACS buffer (PBS containing 2% FBS and 0.01% NaN3). Mice were then euthanized by CO2 asphyxiation or cervical dislocation. Bone marrow was collected by flushing of the femurs. Spleen cells were collected following mechanical disaggregation. Single-cell suspensions prepared from blood, spleen, and bone marrow were subjected to RBC lysis using Ammonium-Chloride-Potassium (ACK) lysis buffer and washed twice before blocking in rabbit immunoglobulin commercially purchased from Sigma-Aldrich (St Louis, MO). Blocked RBC-depleted cells were stained using fluorescently conjugated antibodies to anti-human CD45 antibody (clone HI30) and anti-mouse CD45 antibody (clone A20), from BD Biosciences (San Jose, CA). Additionally, anti-human CD20 antibody (clone 2H7) from BioLegend (San Diego, CA) was used to confirm that the human Daudi cells were CD20 positive. Fixable viability dye eFluor® 780 from BD Biosciences (San Jose, CA) was used to stain dead cells to discriminate viable from non-viable cells during flow cytometric analysis. All phenotyping analyses were carried out by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA), and data were processed using FlowJo software.
Verma M.K., Clemens J., Burzenski L., Sampson S.B., Brehm M.A., Greiner D.L, & Shultz L.D. (2017). A Novel Hemolytic Complement-Sufficient NSG Mouse Model Supports Studies of Complement-Mediated Antitumor Activity In Vivo. Journal of immunological methods, 446, 47-53.
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9

Flow Cytometric Analysis of Germinal Center B Cells

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Spleens were prepared into single cell suspension and red blood cells were lysed
in cold distilled water. After filtered through 70-µm nylon mesh and counting
using a MACSQuant analyzer (Miltenyi Biotec), cells were incubated with anti-CD16/32
antibody (Bio X Cell) to block Fc receptors and stained with fixable viability dye eFluor
780 (eBioscience). Cells were then stained for 20 min in staining media (Hank’s
balanced salt solution, 3% FBS, 0.02% sodium azide, 1 mM EDTA) with
primary antibodies including B220-FITC, B220-eVolve 655 (clone RA3–6B2),
GL7-eFluor 660, GL7-eFluor 450 (clone GL7), CD95-PE, CD95-APC (clone 15A7) (eBioscience),
peanut agglutinin (PNA)-biotin (Vector Laboratories), CD86-Pe-Cy7 (clone GL-1; BioLegend)
or CD184-biotin (2b11/CXCR4; BD Biosciences). Cells stained with biotin-labeled antibodies
were incubated with streptavidin-eFluor 450 (eBioscience). For intracellular staining,
cells were stained with fixable viability dye eFluor 780, permeabilized and fixed using a
cytofix/cytoperm plus kit (BD Biosciences) according to manufacturer suggested protocol.
Cells were then stained with YY1 antibody (H-414; Santa Cruz Biotechnology) and DyLight
594-conjugated secondary antibody (Jackson ImmunoResearch). Flow cytometry analysis was
performed on an LSRII FACS or a FACSAria cell sorter (BD Biosciences), and analyzed using
FlowJo software (FlowJo).
Trabucco S.E., Gerstein R.M, & Zhang H. (2016). YY1 regulates the germinal center reaction by inhibiting apoptosis. Journal of immunology (Baltimore, Md. : 1950), 197(5), 1699-1707.
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10

Isolation of Alveolar Macrophages from Mice

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Mice were either infected or given PBS and euthanized 24 h post-treatment. Lungs were harvested, and dissociated using VDI 12 tissue homogeniser (VWR) in sterile PBS. Single-cell suspensions were obtained by flushing the samples through 70 μm strainer. To exclude dead cells, samples were stained with FVD eFluor 506 (eBioscience), prior to Fc blocking with anti-CD16/CD32 (2.4G2, BD). To sort out CD45+CD11chighSiglecF+ alveolar macrophages, cell suspensions were stained for cell surface proteins using monoclonal antibodies conjugated to allophycocyanin-eFluor 780, allophycocyanin and brilliant violet 421: anti-CD45 (30-F11), anti-CD11c (N418) and anti-SiglecF (E50-2440), purchased from BD Bioscience, eBioscience and Biolegend. RNA from sorted alveolar macrophages was extracted using Power SYBR Green Cells-to-CT Kit (4402954, Ambion) according to manufacturer’s instructions.
Ivin M., Dumigan A., de Vasconcelos F.N., Ebner F., Borroni M., Kavirayani A., Przybyszewska K.N., Ingram R.J., Lienenklaus S., Kalinke U., Stoiber D., Bengoechea J.A, & Kovarik P. (2017). Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection. PLoS Pathogens, 13(11), e1006696.
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