Murine: I-A/I-E-BV421, B220-BV605, F4/80-BV711, Sirpα-PE-Cy7, CD45-FITC, TCR Vβ5.1, 5.2-APC, BV421-CD4, PE-Cy7 CD8a, PerCP-Cy5.5-CD11b, CD40-BV786, H2Kb/H-2Db-Alexa647 (BioLegend); TLR7-PE, CD24-BUV395, CD80-BUV737, F4/80-BV421, CD11b-APC (BD); and eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific). Western blotting: TRIF (Genentech,), MYD88 (Abcam), β-actin (Cell Signaling Technologies). Human: CD64-APC, CD80-BV786 (BD); CD163-FITC, B2M-PE, CD209-APC (DC-SIGN); CD86-PE, CD80-PE-Cy7 (BioLegend); and CD14-PerCP-eFluor 710, CD81-FITC (Invitrogen). LIVE/DEAD Fixable Aqua Dead Cell dye and LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation were from Thermo Fisher Scientific.
Ebioscience fixable viability dye efluor 780
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EBioscience Fixable Viability Dye eFluor 780 is a fluorescent dye used to identify and exclude dead cells in flow cytometry applications. It binds to cellular proteins, allowing for the discrimination of live and dead cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Facsverse, by BD (2 mentions)
Fc gamma receptor block, by BD (2 mentions)
Facsaria 3, by BD (2 mentions)
Cd27 pe cy7, by BD (2 mentions)
Transcription factor buffer set, by BD (2 mentions)
Fcs express v5, by De Novo Software (2 mentions)
Cd14 bv510, by BD (2 mentions)
Cd3 bv510, by BD (2 mentions)
Cd19 bb700, by BD (2 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Cd25 pe cy7, by BD (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Il 2 apc, by BD (2 mentions)
Brefeldin a solution 1000x, by Thermo Fisher Scientific (2 mentions)
Cd69 percp, by BioLegend (2 mentions)
4 1bb bv711, by BioLegend (2 mentions)
Fix perm cell fixation cell permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Ifn γ fitc, by BioLegend (2 mentions)
Tnf a pacific blue, by BioLegend (2 mentions)
45 protocols using ebioscience fixable viability dye efluor 780
1
Murine Immune Cell Phenotyping
2
T Cell Activation Assay
3
Antigen Specificity of Ramos B Cells
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Antigen specificity of (gl-)AR3C and (gl-)HEPC74 Ramos B cells to H77 E2E1 and UKNP4.1.1 E2E1 was detected using labeled probes and flow cytometry as previously42 (link),52 (link). Briefly, biotinylated H77 and UKNP4.1.1 E2E1-foldon-Avi-His were individually multimerized with fluorescently labeled AF647 (Biolegend) and BV421 (Biolegend) streptavidin at a 2:1 protein to fluorochrome molar ratio and incubated for 1 h at 4 °C. Unbound streptavidin conjugates were quenched with 10 µM biotin (Genecopoiea) for 15 min. The antigen-probe cocktails were then used to stain 5 × 105 cells together with live/DEAD dye (eBioscience™ Fixable Viability Dye eFluor™ 780, Thermo Fisher), IgG PE-Cy7 (G18-145, BD Biosciences) in FACS buffer (PBS supplemented with 1 mM EDTA and 2% fetal calf serum). The live/DEAD marker was titrated for signal-to-noise ratio and IgG PE-Cy7 was used in a dilution of 2.5 μL in 50 μL. Stained samples were subsequently washed twice with FACS buffer and acquired on the BD LSRFortessaTM for cell analysis. Analysis was performed using FlowJo v10.8.1. Ramos cells were first gated based on the morphology (FSC-A/SSC-A) and doublets were removed. Live cells were selected and subsequently gated on GFP+ and IgG+. Antigen-specific Ramos B cells were double positive for the HCV H77 and 4.1.1 E2E1- foldon-Avi-His probes (AF647 and BV421).
4
Lymph Node Cell Counting and Phenotyping
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Lymph nodes were transferred to a meshed-cap vial (35 µm mesh), pressed through it with 400 µL of PBS and counted using an automated cell counter (Sysmex, Norderstedt, Germany). Cells were resuspended in FACS buffer (FBS 2%, EDTA 2 mM in PBS) and stained with eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific), followed by incubation with Fc-gamma receptor block (Becton Dickinson). The cells were then stained with fluorochrome-conjugated antibodies: CD3-BV510, CD4-A488 or CD4-APC, CD8-FITC or CD8-BV421 (BioLegend, San Diego, CA, USA). The cells were immediately acquired using FACSVerse (Becton Dickinson) and the data were analyzed with FlowJo software Version 10 (FlowJo_v10.6.1, Ashland, OR, USA). Forward and side scatter gates were used to discriminate doublets and debris (FSC-A, FSC-H, SSC-A × SSC-H). Fluorescence minus one was used as control. Only viable cells were included in the analysis.
5
Evaluating Antibody-Conjugate Effects on Breast and Lung Cancer Cells
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SKBR-3 and A549 cells were seeded in single culture or co-culture in 96-well plates at a density of 8000 cells per well and incubated overnight at 37 °C under a 5% CO2 atmosphere. The incubation media was removed and 100 µL of a 10 nM solution of tested conjugate in DMEM complete medium were added to the plate and incubated for 5 days. Cells were then collected and transferred into a round-bottom 96-well plate suitable for high-throughput flow cytometry. Cells were rinsed 3 times with PBS, resuspended in 50 µL DPBS, and incubated for 30 min at room temperature in the dark with anti-HER2 APC-conjugated antibody (BD Bioscience, Cat#340554) and eBioscience Fixable Viability Dye eFluor 780 (Thermo Scientific, Waltham, MA, USA, Cat#650865-14). Flow cytometry analysis was performed using a BD Fortessa flow cytometer controlled by BD FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (BD Bioscience).
6
Viable Cell Sorting and Preparation for scRNAseq
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Prior to sorting cells, viability staining was performed using eBioscience Fixable Viability Dye eFluor 780 (ThermoFisher Scientific) per manufacturer’s protocol. This was followed by a wash and staining with anti-CD45 PE (BioLegend; San Diego, CA) for 30 min in sorting buffer (0.1% BSA in PBS) at 4 °C. After washing, viable CD45+ and CD45− cells were sorted using the Beckman Coulter MoFlo Astrios. Subsequently, the cells were washed twice and re-suspended in a sorting buffer. A cell number and viability count were performed on a Cellometer Auto 2000 using the ViaStain™ AOPI Staining Solution (Nexcelom Bioscience LLC, Lawrence, MA, USA) immediately prior to scRNAseq.
7
Flow Cytometry Analysis of B Cell Transcription Factors
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PBMC were plated and stimulated with CpG-ODN 2006 (Aurogene Srl) alone or in the presence of 100 μl of CM-hAMSC as described above. After 5 days of culture, cells were collected and protein expression of the transcription factors BCL6, PAX-5, IRF-4, and BLIMP-1 was analyzed by flow cytometry. After exclusion of dead cells by eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific) staining, cells were stained for 20 min at 4°C with CD19 BB700 (SJ25C1, 1:200), CD3 BV510 (UCHT1, 1:100), CD14 BV510 (MΦP9, 1:200), CD27 PE-Cy7 (M-T271, 1:100), all from BD Biosciences. After washing in stain buffer, the cells were fixed and permeabilized with Transcription Factor Buffer Set (BD Biosciences) for 40 min at 4°C, according to the manufacturer's instructions. Then, cells were stained for 40 min at 4°C with specific antibody against BCL6 BV421 (K112-91, 1:30), PAX-5 PE (1H9, 1:100), IRF-4 PE (Q9–343, 1:100), BLIMP-1 Alexa Fluor® 647 (6D3, 1:100). Finally, the cells were washed with Transcription Factor Buffer Set (BD Biosciences), acquired on a FACSAria III (BD Biosciences), and analyzed using FCS express v5.0 (DeNovo Software, Los Angeles, CA, USA).
8
Evaluating PYHIN Proteins' Effects on HIV-1 LTR
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Flow cytometry was used to determine the effect of PYHIN proteins on HIV-1 LTR-driven eGFP expression and cell viability. HEK293T cells were cotransfected with expression constructs for PYHIN proteins co-expressing BFP via an IRES and HIV-1 NL4-3 proviral constructs co-expressing eGFP via an IRES. 48 h after transfection cells were harvested, washed in PBS with 1% FCS and fixed in 2% PFA for 30 min at 4°C. Mean fluorescence intensities (MFI) of eGFP in the BFP+/eGFP+ population and percentage of eGFP+ cells in the BFP+ population (P2/(P2+P1)) was determined. To assess any PYHIN-mediated cytotoxic effect, 48 hours after transfection cells were gently washed in PBS, trypsinized (Pan Biotech, Trypsin/EDTA 0.05%/0.02% in PBS w/o Ca, Mg) and stained with the eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher, #65-0865-14) for 15 minutes at room temperature at dark. Cells were then washed in PBS with 1% FCS and fixed in 2% PFA as previously described and the percentage of dead and living cells was determined on the whole population. Flow cytometric measurements were performed using a BD FACS Canto II flow cytometer.
9
Multi-Parameter Flow Cytometry Analysis of T Cell Subsets
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For flow cytometry analysis, single-cell suspensions from thymus, spleen, lymph nodes, and bone marrows were incubated with diluted eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher) in phosphate-buffered saline (PBS) prior staining with fluorochrome-conjugated antibodies. Intracellular staining was performed after cell fixation with 4% paraformaldehyde (PFA) by incubating the cells with conjugated antibodies in permeabilization buffer (Thermo Fisher Scientific). For the phenotyping of DN subsets, thymocytes were stained with an anti-lineage cocktail (anti-Gr1, anti-CD11b, anti-CD11c, anti-Ter119, anti-CD3, anti-B220, anti-NK1.1, and anti-TCRγδ) and with anti-CD8α and anti-CD4 antibodies. Data acquisition was performed on a BD LSRII flow cytometer and analysis with the FlowJo software.
For DN3 cell purification, thymocytes were first immunomagnetically depleted of CD3-, CD4-, or CD8α-positive cells. Lin-CD44-CD25+CD5- or Lin-CD44-CD25+CD71- DN3 cells were sorted on a BD FACS Aria cell sorter. For peripheral T-cell isolation, total CD4+ T cells and CD8+ T cells were purified from ACK-treated pooled lymph nodes and spleen by magnetic immunodepletion of CD8+, B220+, MHCII+, NK1.1+, Fcγ+, and CD11b+ cells and CD4+, B220+, MHCII+, NK1.1+, Fcγ+, and CD11b+ cells, respectively.
For DN3 cell purification, thymocytes were first immunomagnetically depleted of CD3-, CD4-, or CD8α-positive cells. Lin-CD44-CD25+CD5- or Lin-CD44-CD25+CD71- DN3 cells were sorted on a BD FACS Aria cell sorter. For peripheral T-cell isolation, total CD4+ T cells and CD8+ T cells were purified from ACK-treated pooled lymph nodes and spleen by magnetic immunodepletion of CD8+, B220+, MHCII+, NK1.1+, Fcγ+, and CD11b+ cells and CD4+, B220+, MHCII+, NK1.1+, Fcγ+, and CD11b+ cells, respectively.
10
IFITM Knockdown and ACE2 Expression
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Calu-3 cells were seeded in a 6 well format. The next day, cells were transfected with either non-targeting or IFITM1, 2 or 3 siRNA following the siRNA protocol described above. In the indicated conditions, cells were simulated with 1000 U/ml IFN-β. 6h after the second transfection, cells were harvested using Versene (Thermo Fischer) 15 minutes at 37°C. Staining was performed as previously described.55 (link) In brief, after washing with PBS cells were stained with the eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher) for 15 minutes at room temperature in the dark. Afterwards cells were permeabilized using 200μl of ice-cold methanol for 10 minutes, washed once with FACS buffer (2% FCS in PBS) and blocked with 200μl of FACS buffer on ice for 10 minutes. Then, cells were stained with primary antibody against ACE2 diluted 1:200 in FACS buffer and incubated at 4°C for 45 minutes. Cells were washed twice with FACS buffer and stained with secondary antibody (Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™647) diluted 1:200 in FACS buffer at 4°C. After 30 minutes, cells were washed twice with FACS buffer and resuspended in 100μl of PBS and analysed on BD FACS Canto II using BD FACS Diva and FlowJo software.
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