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Live dead staining

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Live/Dead staining is a technique used to assess cell viability. It utilizes fluorescent dyes that differentiate between live and dead cells, allowing for the quantification of cell populations. This method provides a quick and reliable way to evaluate the health and integrity of cells in various biological samples.

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Lab products found in correlation

Facsdiva 6, by BD (3 mentions) Spe confocal microscope, by Leica (3 mentions) Ic fixation buffer, by Thermo Fisher Scientific (3 mentions) Polystyrene beads, by Merck Group (3 mentions) Mouse serum, by Bio-Rad (3 mentions) Facscanto 2, by BD (3 mentions) Ionomycin, by Merck Group (3 mentions) Cytofix cytoperm kit, by BD (3 mentions) Vico confocal, by Nikon (2 mentions) Calcein am, by Thermo Fisher Scientific (2 mentions) Celltiter blue, by Promega (2 mentions) Facscanto flow cytometer, by BD (2 mentions) Inverted fluorescence microscope, by Olympus (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions) Brefeldin a, by Thermo Fisher Scientific (2 mentions) Lsrfortessa, by BD (2 mentions) Veritas microplate luminometer, by Promega (2 mentions) Tcs lsi macro confocal microscope, by Leica (2 mentions) 96 well plate, by Thermo Fisher Scientific (2 mentions) Triton x 100 lysis buffer, by Merck Group (2 mentions)

Close spelling variants of this product

Live/Dead staining kit (120 citations) Live/Dead cell staining kit (30 citations) Live/Dead Fixable Dead Cell staining kit (11 citations) LIVE/DEAD Aqua staining (10 citations) Aqua Live/Dead fixable staining reagent (7 citations) Live/dead staining solution (7 citations) LIVE/DEAD Fixable Violet Dead Cell staining kit (6 citations) LIVE/DEAD fixable blue dead-cell staining kit (5 citations) Pac-Orange Live/Dead fixable dead staining dye (4 citations) LIVE/DEAD Fixable Violet Dead Staining Kit (4 citations)

92 protocols using live dead staining

1

Live/Dead Cell Staining Protocol

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The following reagents and kits were used in this experiment: live/dead staining (#R37601, Invitrogen, Waltham, MA, USA), RPMI 1640 medium 1× (Cat. N.: SH30027.01, Cytiva, Tokyo, Japan), DPBS (Dulbecco’s phosphate buffered saline, ref. no. 21-031-CV, Corning Inc., Corning, NY, USA), DMEM/F-12 50/50, 1× (Dulbecco’s mod. eagle’s medium/Ham’s F-12 50/50 Mix, without L-glutamine, Corning, ref. no. 15-090-CV), FBS (fetal bovine serum, Cat. No.: S11150, Atlanta Biologicals®, Flowery Branch, GA, USA), pen/strep (penicillin–streptomycin, 10,000 units/mL penicillin, 10,000 µg/mL streptomycin, gibco®, ref. no. 15140-122), insulin (recombinant human insulin, gibco®, formula no. A11382IJ), UCG (Ultroser™ G, serum substitute for animal cell culture, PALL Life Sciences, 20 mL, ref. no. 15950-017), Exoview Kit, Avidin/Biotin Blocking Kit (SP-2001, Vector Labs, Burlingame, CA, USA), VECTASTAIN® ABC-HRP Kit, peroxidase (Vector Labs, PK-4000), DAB Substrate Kit, peroxidase (HRP), and nickel, (3,3′-diaminobenzidine) (Vector Labs, SK-4100).
Zha D., Rayamajhi S., Sipes J., Russo A., Pathak H.B., Li K., Sardiu M.E., Bantis L.E., Mitra A., Puri R.V., Trinidad C.V., Cain B.P., Isenberg B.C., Coppeta J., MacLaughlan S., Godwin A.K, & Burdette J.E. (2023). Proteomic Profiling of Fallopian Tube-Derived Extracellular Vesicles Using a Microfluidic Tissue-on-Chip System. Bioengineering, 10(4), 423.
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2

Evaluating IL-27 Effects on T Cell Activation

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To study the effect of IL-27 in T cells a time course stimulation assay was performed to evaluate the upregulation of CD69 expression in T cells incubated with rhIL-27 at concentration: 0.5ng/mL, 5ng/mL, 25ng/mL and 100ng/mL and media as control for overnight or 6 hours (Figure S3C). PBMCs were thawed with X-Vivo media (Lonza, MD) containing Benzonase nuclease (50 U/mL; Millipore Sigma, MO) and cultured in the absence or presence of rhIL-27 (100 ng/mL). After overnight culture, PBMCs from HIV infected participants (n = 17, Table S1) were stimulated with HIVGag peptide pool (2 μg/mL, NIH AIDS Reagent Program), and PBMCs from healthy donors (n = 17) were stimulated with the CMV, EBV and Influenza (CEF) peptide pools (5μg/mL, NIH AIDS Reagent Program). DMSO was used as control in the unstimulated culture condition. After 2 hours of stimulation, Brefeldin A (10 μg/mL, Calbiochem, CA) was added and cultured for additional 4 hours. Cells were harvested and stained with Live/Dead staining (Invitrogen, CA). Cells were incubated with human IgG (10 μg/mL, Sigma, MO) to block Fc receptors followed by a cocktail of mAbs as described in Table S2, Panel 5.
Cheng J., Myers T.G., Levinger C., Kumar P., Kumar J., Goshu B.A., Bosque A, & Catalfamo M. (2021). IL-27 induces IFN/STAT1-dependent genes and enhances function of TIGIT+ HIVGag-specific T cells. iScience, 25(1), 103588.
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3

Expansion and Phenotyping of Activated T Cells

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T cells were isolated from the non-adherent fraction of the osteoclast differentiation cultures and activated with CD3/CD28 mAbs coated beads (T cell TransAct™, Miltenyi Biotech, Auburn, CA). After 3 days of stimulation, IL-2 (50 U/ml, TECIN™, National Cancer Institute, Frederick, MD) was added to the media. At day 6, the phenotype of the expanded T cells was analyzed by flow cytometry. Prior to surface staining, T cells were stained with LIVE/DEAD staining (Invitrogen, MA), followed by incubation with 1 μg/ml human IgG (Sigma, MO) to block Fc receptors. Cell surface staining were performed using a cocktail of mAbs: CD3 (clone UCHT1) and CD8 (clone RPA-T8) both from BD Biosciences, CA. Cells were acquired using a BD FACS Symphony flow cytometer and analyzed using FlowJo.
Li T., Hadigan C., Whitlock J.M., Qin J., Kumar J., Kumar P, & Catalfamo M. (2022). IL-27 Modulates the Cytokine Secretion in the T Cell–Osteoclast Crosstalk During HIV Infection. Frontiers in Immunology, 13, 818677.
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4

Live/Dead Cell Viability Analysis

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To evaluate cell viability, we used the Live/Dead staining (Invitrogen); 500 μL of a solution consisting of 1.5 ml of Phosphate Buffered Saline (PBS), 3 μL of EthD‐1 and 1.5 μL of calcein, was added to 3D constructs. Samples were incubated for 45 min in the dark, then the solution was removed, and cell nuclei were counterstained with 500 μL 4′,6‐diamidino‐2‐phenylindole (DAPI) for 10 min according to the protocol. Fluorescent image acquisition was carried out by semi‐confocal microscope (ViCo confocal, Nikon).
Viability and differentiation tests were performed as well as morphological and gene expression analysis at six different time points (1, 4, 7, 14, 21, and 28 days in culture).
Ronzoni F.L., Aliberti F., Scocozza F., Benedetti L., Auricchio F., Sampaolesi M., Cusella G., Redwan I.N., Ceccarelli G, & Conti M. (2022). Myoblast 3D bioprinting to burst in vitro skeletal muscle differentiation. Journal of Tissue Engineering and Regenerative Medicine, 16(5), 484-495.
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5

Live/Dead Cell Viability Assay

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LIVE/DEAD® staining (Cell viability®, Invitrogen) was performed to assess viability of hASCs on the solid constructs 1, 3 and 7 days after seeding. 300μL of PBS containing 4 μM EthD-1 and 2 μM Calcein-AM (Invitrogen) was added to each sample followed by incubation at room temperature for 10 min. The samples were then imaged using a fluorescent microscope (Zeiss SteREO Lumar.V12 fluorescence stereomicroscope) to detect live (green) and dead (red) cells on the samples. Cells cultured on 2D were served as positive control.
Forghani A., Garber L., Chen C., Tavangarian F., Tighe T.B., Devireddy R., Pojman J.A, & Hayes D. (2018). Fabrication and Characterization of Thiol- Triacrylate Polymer via Michael Addition Reaction for Biomedical Applications. Biomedical materials (Bristol, England), 14(1), 015001.
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6

ILC Function Assay Protocol

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For ILC function assays, spleen cells were stimulated for 4 h with plate bound anti-NK1.1 in the presence of 1 × protein transport inhibitor cocktail (PTIC) containing Brefeldin A/Monensin (eBioscience, Thermo Fisher Scientific) and anti-CD107a (eBio1D4B, eBioscience, Thermo Fisher Scientific) in complete Iscove's DMEM medium (Corning). Cells were incubated during stimulation at 37°C in 5% CO2 for 4 h. Cells were then first surface stained then intracellularly stained to measure function. ILC phenotypes were measured directly ex vivo. Spleen cells were stained following procedures indicated below after fixable Live/Dead staining (Invitrogen).
Ivanova D.L., Krempels R., Denton S.L., Fettel K.D., Saltz G.M., Rach D., Fatima R., Mundhenke T., Materi J., Dunay I.R, & Gigley J.P. (2020). NK Cells Negatively Regulate CD8 T Cells to Promote Immune Exhaustion and Chronic Toxoplasma gondii Infection. Frontiers in Cellular and Infection Microbiology, 10, 313.
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7

Evaluating Cytotoxicity of PA-RGDS Hydrogel for Cardiomyocytes

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In order to examine if PA-RGDS is cytotoxic to CMs, we performed Live/Dead staining (Invitrogen) for CMs cultured in PA-RGD gel. A total of 5 × 105 freshly isolated and cultured NRCMs contained in 50 μL of advanced DMEM media with 3% FBS were mixed with 50 μL of the PA-RGDS solution followed by the addition of 14 μL of CaCl2 (0.1 M) solution to initiate gel polymerization. Subsequently, the CM/PA-RGDS constructs were cultured in CM culture medium containing advanced DMEM/F12 supplemented with 3% FBS and ITS for 7 days and harvested for Live/Dead staining. To show the live cell distribution, confocal images (z-stacks) were captured at either the center or the edge of CM/PA-RGDS constructs using a Zeiss LSM 510 Meta confocal laser scanning microscope and LSM 510 Image software (CLSM, Carl Zeiss). Quantification of live or dead cells was performed using ImageJ software.
Ban K., Park H.J., Kim S., Andukuri A., Cho K.W., Hwang J.W., Cha H.J., Kim S.Y., Kim W.S., Jun H.W, & Yoon Y.S. (2014). Cell Therapy with Embryonic Stem Cell-Derived Cardiomyocytes Encapsulated in Injectable Nanomatrix Gel Enhances Cell Engraftment and Promotes Cardiac Repair. ACS Nano, 8(10), 10815-10825.
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8

Characterizing Stem Cell Viability and Proliferation

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Acute cell viability following extrusion was characterized by LIVE/DEAD staining (Invitrogen), following the manufacturer’s instructions (n = 4). Cell sedimentation was performed as previously described [23 (link)]. Briefly, 70 μL of bioink containing NPCs were mixed with 4 μM calcein AM and added to a 70 μL microcuvette (BrandTech) and incubated at 37 °C for 1 h (n = 3). Following incubation, the cuvette was quickly turned on its side and imaged using a confocal microscope. To characterize the degree of cell proliferation, NPC-containing alginate constructs were manually transferred to a lysis buffer of 20 mM Tris HCl (ThermoFisher Scientific), 150 mM NaCl (ThermoFisher Scientific), 0.5% Triton X-100 (Sigma-Aldrich), in DPBS, pH 7.4, and disrupted by sonication. Total DNA content was quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) (n = 4), following the manufacturer’s instructions, and normalized to day 0 controls that were collected 30 min post-printing. Metabolic activity of expansion lattices was quantified using CellTiter Blue (Promega), following the manufacturer’s instructions (n = 4). Metabolic activity was quantified daily for 30 min and normalized to the day 1 results. Immediately following quantification, expansion lattices were rinsed in warm Neurobasal-A Medium and Stemness Maintenance Medium was added.
Lindsay C.D., Roth J.G., LeSavage B.L, & Heilshorn S.C. (2019). Bioprinting of stem cell expansion lattices. Acta biomaterialia, 95, 225-235.
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9

Determination of EBV Infectious Titers

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Viral titers were determined from supernatants of 293/EBV-wt cells producing EBV throughout 3 days. In order to estimate infectious units in supernatants of EBV producing cells, the supernatant was filtered through 0.45 μm filters and inoculated on Raji or Ramos cells (4 × 104) for 48 h as indicated. Titrations were always performed in duplicates. Cells were analyzed by flow cytometry using a BD FACS Canto Flow Cytometer and the FlowJo software. Dead cells were excluded through Live/Dead staining (Invitrogen) and EBV infected Raji cells were detected in the GFP-positive gate.
Nowag H., Guhl B., Thriene K., Romao S., Ziegler U., Dengjel J, & Münz C. (2014). Macroautophagy Proteins Assist Epstein Barr Virus Production and Get Incorporated Into the Virus Particles. EBioMedicine, 1(2-3), 116-125.
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10

Quantifying Airway Epithelial Cell Populations

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Differentiated human airway epithelia were incubated with fixable Live/Dead staining (Invitrogen) for 15 minutes before washing twice with PBS. Then, cultures were incubated with Accutase cell dissociation solution (Innovative Cell Technologies, Inc.) for 15 minutes at 37°C. Single-cell suspensions were collected and centrifuged at 250g for 10 minutes. GFP+ or mCherry+ cells could be quantified directly by flow cytometry. For antibody staining, cells were fixed with 4% paraformaldehyde for 30 minutes, and then permeabilized using the eBioscience FOXP3/Transcription factor staining buffer set (Invitrogen). Cells were blocked overnight at 4°C with 10% normal goat serum flow buffer. Antibodies were diluted in 10% flow buffer and incubated for 1 hour at room temperature. Antibodies included PE/Dazzle 594 NGFR/CD271 (catalog 345120, BioLegend, mouse monoclonal antibody; 1:500), P63-488 (catalog NBP3-08736AF488, Novus, rabbit monoclonal antibody; 1:1000), and BSND 568 (catalog Ab196017, Abcam, rabbit polyclonal antibody; 1:1000). After incubation with primary antibodies, cells were washed 3 times with flow buffer and resuspend in 200 μL flow buffer. Data were collected using an Attune NxT flow cytometer (Invitrogen) and analyzed by FlowJo software. Example plots are shown in Supplemental Figures 1–4.
Lei L., Traore S., Romano Ibarra G.S., Karp P.H., Rehman T., Meyerholz D.K., Zabner J., Stoltz D.A., Sinn P.L., Welsh M.J., McCray PB J.r, & Thornell I.M. (2023). CFTR-rich ionocytes mediate chloride absorption across airway epithelia. The Journal of Clinical Investigation, 133(20), e171268.
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