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Fixable viability dye conjugated with efluor780 fluorochrome

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Fixable Viability Dye conjugated with eFluor780 fluorochrome is a fluorescent label used to identify and distinguish live and dead cells in flow cytometry applications. It binds to proteins with amine groups, which are present on the surface of all cells, allowing for the detection of cell viability.

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Lab products found in correlation

Cytoflex instrument, by Beckman Coulter (2 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions) Lsm 700, by Zeiss (1 mentions) Cytexpert software, by Beckman Coulter (1 mentions) Pharm lyse solution, by BD (1 mentions) Anti cd8α buv805 clone 53 6, by BD (1 mentions) Cell fix solution, by BD (1 mentions) β glycerophosphate, by Merck Group (1 mentions) Alizarin red staining, by Merck Group (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)

4 protocols using fixable viability dye conjugated with efluor780 fluorochrome

1

Analyzing hNPC Viability in Alginate Beads

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hNPCs in alginate beads (n = 5) were incubated for 10 min at 4°C in 55 mmol/L sodium citrate, 30 mmol/L EDTA, 0.15 M NaCl, pH 6.8 to dissolve beads and analyze cell count and viability using a CytoFLEX instrument with CytExpert Software (v.2.1, Beckman Coulter). Cell count was assessed at 1, 4, 10, and 14 days and expressed as event/μL. To evaluate the effect of EV treatment on cell death, hNPCs (n = 5) were stained with Fixable Viability Dye conjugated with eFluor780 fluorochrome (Affymetrix eBioscience, Thermo Fisher Scientific) after 24 h of treatment, using the gating strategy depicted in Figure 1. Similarly, cell viability was determined by analyzing membrane integrity with the LIVE/DEAD assay, following the manufacturer's instructions. Briefly, hNPCs in alginate beads (n = 5) were incubated for 45 min with ethidium homodimer‐2 and calcein acetoxymethylester (AM) at room temperature, washed three times with PBS and incubated with Hoechst 33258 (Life Technologies) to stain the nuclei. After incubation, green, red, and blue fluorescence were detected using a confocal laser scanning microscope (Zeiss LSM700, Carl Zeiss).
26 (link)
Tilotta V., Vadalà G., Ambrosio L., Di Giacomo G., Cicione C., Russo F., Darinskas A., Papalia R, & Denaro V. (2023). Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate‐bead culture model. JOR Spine, 7(1), e1274.
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2

Quantification of Gag-specific CD8+ T Cells

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Spleen and LN cells were incubated with Fixable Viability Dye conjugated with eFluor780 fluorochrome (Affymetrix, eBioscience, Santa Clara, CA, USA), and background staining was blocked with anti‐FcγR mAb (clone 2.4G2). Cells were then incubated for 15 minutes at 4°C with H‐2k(d) AMQMLKETI APC‐labelled Tetramer (Tetr‐gag, NIH Tetramer Core Facility, Atlanta, GA) and PE‐labelled Pentamer (Pent‐gag, Proimmune, Oxford, UK) to stain for gag197‐205(gag)‐specific CD8 T cells. Cells were incubated for further 15 minutes at 4°C after addition of the following mAbs: anti‐CD3 peridinin chlorophyll protein (PerCP)‐Cy5.5 (clone 145‐2C11; BD Biosciences, San Jose, CA, USA), anti‐CD8α BUV805 (clone 53‐6.7, BD Biosciences) and anti‐CD62L phycoerythrin (PE)‐Cy7 (clone MEL‐14, Biolegend, San Diego, CA, USA). Blood samples were incubated for 30 minutes at RT with the above antibodies/reagents that were placed all together. After washing, blood cells were fixed with Cell Fix solution (BD Biosciences). Red cells were lysed with Pharm Lyse solution (BD Biosciences).
Simonetti S., Natalini A., Folgori A., Capone S., Nicosia A., Santoni A, & Di Rosa F. (2019). Antigen‐specific CD8 T cells in cell cycle circulate in the blood after vaccination. Scandinavian Journal of Immunology, 89(2), e12735.
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3

Osteoblast Differentiation and Viability Assay

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Human primary OBs were obtained from bone marrow samples of healthy patients undergoing total hip replacement at Policlinico Campus Bio-Medico. The procedure was approved by the Ethical Committee of the Campus Bio-Medico University of Rome and informed consent from patients was collected in accordance with the Declaration of Helsinki principles (Prot 21/15 OSS). Bone marrow mesenchymal stem cells (BM-MSCs) were isolated as previously described [34 (link)]. OB differentiation was achieved by culturing BM-MSCs at an initial density of 5 × 104 in 24-well plates while adding 10 mM beta-glycerophosphate (Sigma Aldrich, St. Louis, MO, USA), 50 μM ascorbic acid (Sigma Aldrich, St. Louis, MO, USA), and 100 nM dexamethasone (Sigma Aldrich, St. Louis, MO, USA) to the culture medium for 28 days. OBs were treated with ribociclib, palbociclib, and abemaciclib for 7 days (from day 21 to day 28). OB viability was evaluated by flow cytometry using Fixable Viability Dye conjugated with eFluor780 fluorochrome (eBioscience-Thermo Fisher Scientific, Waltham, MA, USA). OB activity was analyzed by Alizarin Red staining (Sigma Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions.
Iuliani M., Simonetti S., Ribelli G., Napolitano A., Longo U.G., Vincenzi B., Orsaria P., Denaro V., Tonini G., Santini D, & Francesco P. (2022). Biological Effects of Cyclin-Dependent Kinase Inhibitors Ribociclib, Palbociclib and Abemaciclib on Breast Cancer Bone Microenvironment. International Journal of Molecular Sciences, 23(5), 2477.
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4

Cell Cycle Analysis by Flow Cytometry

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The cell cycle was analyzed using the following gaiting strategy (Figure 6) [35 (link)]. Briefly, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience-Thermo Fisher Scientific, Waltham, MA, USA) for intracellular staining with anti-Pan Cytokeratin Alexa Fluor 488 (clone AE1/AE3 eBioscience-Thermo Fisher Scientific, Waltham, MA, USA), anti-Ki67-APC (clone 20Raj1 eBioscience-Thermo Fisher Scientific, Waltham, MA, USA), and a Propidium Iodure (PI) solution (50 μg/mL PI+ 40 ng/mL RNAseA+ 0.1% of Triton) ((Sigma Aldrich, St. Louis, MO, USA). Dead cell exclusion was performed with Fixable Viability Dye conjugated with eFluor780 fluorochrome (eBioscience-Thermo Fisher Scientific, Waltham, MA, USA). Samples were analyzed by CytoFlex instrument (Beckman Coulter, Brea, CA, USA) using the CytExpert Software, v.2.1.
Iuliani M., Simonetti S., Ribelli G., Napolitano A., Longo U.G., Vincenzi B., Orsaria P., Denaro V., Tonini G., Santini D, & Francesco P. (2022). Biological Effects of Cyclin-Dependent Kinase Inhibitors Ribociclib, Palbociclib and Abemaciclib on Breast Cancer Bone Microenvironment. International Journal of Molecular Sciences, 23(5), 2477.
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