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Violet fluorescent reactive dye

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The Violet fluorescent reactive dye is a fluorescent labeling agent that can be used to detect and visualize target molecules in various research and diagnostic applications. It emits light in the violet spectrum when excited by the appropriate wavelength of light.

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Lab products found in correlation

Golgiplug, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Aqua fluorescent reactive dye, by Thermo Fisher Scientific (2 mentions) Fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions) X20 analyzer, by BD (2 mentions) Qdot 605, by Thermo Fisher Scientific (1 mentions) Qdot 655, by Thermo Fisher Scientific (1 mentions) Annexin 5 fitc, by BD (1 mentions) Anti pd 1 fitc, by Thermo Fisher Scientific (1 mentions) Anti bcl2 fitc, by BD (1 mentions) Perm wash buffer, by BD (1 mentions) Cytofix cytoperm reagent, by BD (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Granzyme b apc, by BD (1 mentions) Ifn γ fitc, by BD (1 mentions) Saponin, by Merck Group (1 mentions) Formaldehyde, by Polysciences (1 mentions) Pe cy7 tnfα, by BD (1 mentions) Ebioscience protein transport inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Biosciences lsrii flow cytometer, by BD (1 mentions)

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Fluorescent dyes (25 citations) (fluorescent violet reactive, (3 citations) Violet fluorescent reactive dye VVD (2 citations)

7 protocols using violet fluorescent reactive dye

1

Intracellular TNFα Quantification

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Cells were seeded into non-treated tissue culture 24-well plates or round-bottom 96-well plates. The next day cells were stimulated with the indicated TLR ligands. To measure TNFα production, BrefeldinA (BD GolgiPlug, BD Biosciences) was added to cells 30 min after stimulation, and cells were collected after an additional 5.5 h. Dead cells were excluded using a fixable live/dead stain (Violet fluorescent reactive dye, Invitrogen). Cells were stained for intracellular TNFα with a Fixation & Permeabilization kit according to manufacturer’s instructions (eBioscience).
For flow cytometry on mouse cells, dead cells were excluded using a fixable live/dead stain (Aqua fluorescent reactive dye, Invitrogen) or DAPI and all stains were carried out in PBS containing 1% BSA (w/v) and 0.1% Azide (w/v) including anti-CD16/32 blocking antibody. Cells were stained for 20 min at 4°C with surface antibodies. Data were acquired on a LSRFortessa or X20 analyzer (BD Biosciences). See Extended Data Fig 10 for gating strategies.
Majer O., Liu B., Kreuk L.S., Krogan N, & Barton G.M. (2019). Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity. Nature, 575(7782), 366-370.
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2

Multiparametric Flow Cytometry Analysis

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The cell culture medium was RPMI 1640 from HyClone (Logan, UT), supplemented with 10% heat inactivated fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin. Fluorochrome-conjugated antibodies used to study cell phenotype, differentiation and other expressions with flow cytometry were anti-CD3–cyanine 7 (Cy7) allophycocyanin (APC), anti-Ki67-fluorescein isothiocyanate (FITC), anti-Bcl2-FITC, and Annexin V-FITC that were purchased from BD Biosciences (San Jose, CA). Unconjugated antibodies to CD4, CD8 and CD62L were purchased from Harlan (Indianapolis, IN) and were conjugated with Qdot605, Qdot655 and Alexa Fluor 688 from Invitrogen (Carlsbad, CA). The in-house conjugates were validated by comparison with commercial conjugates. Anti-PD-1-FITC, anti-CD272-phycoerythrin (PE), anti-CD152-PE, anti-Tim1-biotin, anti-Tim3-biotin, anti-CD223-PE and anti-CD127-Cy7PE were purchased from eBioscience Inc (San Diego, CA). Streptavidin (SA)-PE was purchased from ProZyme (Hayward, CA). Violet fluorescent reactive dye (Invitrogen, CA), anti-CD19- and anti-CD16/32-Pacific blue (eBioscience) were used as cell viability and lineage markers to exclude dead and non-T cells from analysis. Fluorochrome-conjugated epitope peptide–MHC class I complexes (pMHC), Db-M187-195-APC (DbM187-APC) and Kd-M282-90-PE (KdM282-PE), were purchased from Beckman Coulter (Fullerton, CA).
Liu J., Haddad E.K., Marceau J., Morabito K.M., Rao S.S., Filali-Mouhim A., Sekaly R.P, & Graham B.S. (2016). A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology. PLoS Pathogens, 12(3), e1005486.
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3

Intracellular TNFα Quantification

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Cells were seeded into non-treated tissue culture 24-well plates or round-bottom 96-well plates. The next day cells were stimulated with the indicated TLR ligands. To measure TNFα production, BrefeldinA (BD GolgiPlug, BD Biosciences) was added to cells 30 min after stimulation, and cells were collected after an additional 5.5 h. Dead cells were excluded using a fixable live/dead stain (Violet fluorescent reactive dye, Invitrogen). Cells were stained for intracellular TNFα with a Fixation & Permeabilization kit according to manufacturer’s instructions (eBioscience).
For flow cytometry on mouse cells, dead cells were excluded using a fixable live/dead stain (Aqua fluorescent reactive dye, Invitrogen) or DAPI and all stains were carried out in PBS containing 1% BSA (w/v) and 0.1% Azide (w/v) including anti-CD16/32 blocking antibody. Cells were stained for 20 min at 4°C with surface antibodies. Data were acquired on a LSRFortessa or X20 analyzer (BD Biosciences). See Extended Data Fig 10 for gating strategies.
Majer O., Liu B., Kreuk L.S., Krogan N, & Barton G.M. (2019). Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity. Nature, 575(7782), 366-370.
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4

Multiparametric Immune Response Assay

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PBMCs were isolated and (1-2 x 106) were resuspended in 150 µl of complete medium containing 0.2 µg of each costimulatory antibody CD28 and CD49b. Stimulation was performed in 96 well/plates using 2 µM of each peptide pool or SEB (as positive control) or medium alone (as negative control). Brefeldin A was added to each well at a final concentration of 10 µg/ml and the plate was incubated at 37°C, 5% CO2 overnight. The cells were then washed, stained with a viability dye (violet, fluorescent reactive dye, Invitrogen), fixed and permeabilized with the BD Cytofix/Cytoperm reagent. Permeabilized cell samples were then stored at -80°C before the staining procedure. Antibody staining was performed in a single step following permeabilization. All the used antibodies are listed in Table 1. After 30 min of incubation on ice in the dark, cells were washed in BD Perm/Wash buffer. Cells were counted with an LSR II (BD) immediately after the staining procedure and FlowJo software was used for data analysis.
Lorenzen E., Contreras V., Olsen A.W., Andersen P., Desjardins D., Rosenkrands I., Juel H.B., Delache B., Langlois S., Delaugerre C., Joubert C., Dereuddre-Bosquet N., Bébéar C., De Barbeyrac B., Touati A., McKay P.F., Shattock R.J., Le Grand R., Follmann F, & Dietrich J. (2022). Multi-component prime-boost Chlamydia trachomatis vaccination regimes induce antibody and T cell responses and accelerate clearance of infection in a non-human primate model. Frontiers in Immunology, 13, 1057375.
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5

Multiparametric Intracellular Immune Profiling

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Fluorescent intracellular staining was performed as previously described [28 (link)]. In brief, 2 × 106 splenocytes were stained with APC-Cy7-CD3, Alexa Fluor700-CD8a, and CD16/32 Fc-block antibodies, fixed using 2% formaldehyde (Polysciences, Warrington, PA), permeabilized using 0.2% saponin (Sigma-Aldrich, St. Louis, MO), stained for intracellular cytokines using APC-granzyme B, FITC-IFNγ, PE-perforin, PE-Cy7-TNFα (BD Biosciences, San Diego, CA), and PerCpCy5.5-IL-2 (BioLegend, San Diego, CA), and stained for dead cell exclusion using violet fluorescent reactive dye (Invitrogen, Carlsbad, CA). Cells were analyzed and 2 × 106 events per sample were captured with a LSRII flow cytometer (BD Biosciences) using FlowJo software (Tree Star, San Carlos, CA).
Quiroga D., Aldhamen Y.A., Appledorn D.M., Godbehere S, & Amalfitano A. (2015). Strengthened tumor antigen immune recognition by inclusion of a recombinant Eimeria antigen in therapeutic cancer vaccination. Cancer immunology, immunotherapy : CII, 64(4), 479-491.
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6

Multiparametric Analysis of Splenocyte Responses

Cited 1 time
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Two million splenocytes were cultured for 5–6 hours with the peptide pools used above, as previously described [44 (link)], and with eBioscience protein transport inhibitor cocktail (Invitrogen). Surface (for CD4 and CD8) and intracellular (for remaining markers) staining followed. Biolegend anti-mouse antibodies conjugated to fluorophores used in this experiment included CD3ε-PE/Cy5 (145-2C11), CD4-FITC (RM4-5), CD8a-APC/Cy7 (53-6.7), IFNγ-APC (XMG1.2), TNFα-BV605 (MP6-XT22), and IL-2-PE-Cy7 (JES6-5H4). Live-dead exclusion was performed using violet fluorescent reactive dye (Invitrogen). Data was collected using a BD Biosciences LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo v10 (FlowJo LLC, Ashland, OR, USA).
Wojtak K., Perales-Puchalt A, & Weiner D.B. (2019). Novel Synthetic DNA Immunogens Targeting Latent Expressed Antigens of Epstein–Barr Virus Elicit Potent Cellular Responses and Inhibit Tumor Growth. Vaccines, 7(2), 44.
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7

Expression of Cell Surface Markers

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To determine the expression of CD44, CD24, and CD49f on the cell surface of 231, 231.Br, and 231.Br.HER2 cells, we performed flow cytometry analysis. Cells were washed twice with phosphatase buffered saline (PBS) solution and then harvested with versene/0.48 mM EDTA (Invitrogen). Detached cells were washed with PBS supplemented with 0.5% FBS (stain buffer), centrifuged at 1200 rpm for 5 min, and resuspended in stain buffer. A single-cell suspension was incubated with APC-conjugated anti-CD44 (1:20) and PE-conjugated anti-CD24 (1:10), or with PerCP-Cy7-conjugated anti-CD49f (1:20). All antibodies were purchased from BD Biosciences (Temse, Belgium). Primary antibodies or the respective isotype controls (BD Biosciences) were incubated at 4 °C in the dark for 20 min. A cell viability marker was included (1:100 dilution, violet fluorescent reactive dye, Invitrogen) to discriminate dead cells. Cells were analyzed using a BD FACS Canto-II flow cytometer (BD Biosciences, Temse, Belgium).
Dionísio M.R., Vieira A.F., Carvalho R., Conde I., Oliveira M., Gomes M., Pinto M.T., Pereira P., Pimentel J., Souza C., Marques M.M., Duval da Silva V., Barroso A., Preto D., Cameselle-Teijeiro J.F., Schmitt F., Ribeiro A.S, & Paredes J. (2020). BR-BCSC Signature: The Cancer Stem Cell Profile Enriched in Brain Metastases that Predicts a Worse Prognosis in Lymph Node-Positive Breast Cancer. Cells, 9(11), 2442.
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