Trypan blue

Manufactured by Avantor

Sourced in United States, United Kingdom

Trypan blue is a dye used in cell counting and cell viability assays. It is a vital stain, meaning it only penetrates cells with damaged membranes, allowing live (unstained) and dead (stained) cells to be distinguished and counted.

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Lab products found in correlation

Ammonium chloride, by STEMCELL (7 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) Streptomycin, by Thermo Fisher Scientific (3 mentions) Nitrocellulose membrane, by Avantor (2 mentions) Ero1 lα, by Cell Signaling Technology (2 mentions) Sheep anti mouse igg hrp linked polyclonal antibody, by Avantor (2 mentions) Donkey anti rabbit igg hrp linked polyclonal antibody, by Avantor (2 mentions) Ezblock protease inhibitor cocktail, by Avantor (2 mentions) Atf 6 65 880 s perk 5683 s phospho perk thr980 3179 s ire 1α 3294 s bip 3183 s chop 5554 s pdi, by Cell Signaling Technology (2 mentions) β actin a5441 0.5 ml antibody, by Merck Group (2 mentions) Phospho ire1α antibody ser724, by Thermo Fisher Scientific (2 mentions) 17 dmag, by Avantor (2 mentions) Ripa buffer aaj63306 ap, by Avantor (2 mentions) Fetal calf serum (fcs), by Avantor (2 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) 2 7 dichlorofluorescein diacetate, by Merck Group (2 mentions) Hepg2, by American Type Culture Collection (2 mentions)

Close spelling variants of this product

Trypan blue solution (10 citations) Trypan blue dye (5 citations) Trypan blue exclusion assay (2 citations)

41 protocols using trypan blue

Cell Viability and Proliferation Assay

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For direct cell number detection, cell were detached at indicated day by 0.1% Trypsin/EDTA solution (Invitrogen), and washed with PBS. Cell suspensions were mixed with an equal volume of 0.4% trypan blue (VWR, Radnor, PA), and viable cells (trypan blue negative cells) were counted. In some experiments, cell proliferation/viability was measured by an MTT assay (BMR Service, Buffalo, NY) according to manufacturer’s instruction. In brief, cell medium was aspirated and then 0.3 ml MTT working solution was added into 24 well. After 30 min incubation at 37oC, MTT solution was aspirated, and cells were incubated with 0.3 ml DMSO for 2 min. The DMSO extracts were transferred to a 96-well plate and absorbance was measured with micro-plate reader at a wavelength of 540 nm.

So E.Y, & Ouchi T. (2014). BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction. BMC Cancer, 14, 548.

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Comprehensive Cell Viability Assays for D. discoideum

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Cell viability was examined by trypan blue (VWR), propidium iodide (BD Biosciences), and Annexin V staining (BD Biosciences). For Annexin V staining, cells were washed twice with cold PBS and suspended in 1X Binding Buffer (10 mM Hepes pH 7.4, 140 mM NaCl, and 2.5 mM CaCl₂) at a concentration of 1 million cells/ml. One hundred microliters of the solution was transferred to a new tube and 5 μl of FITC Annexin V was added to the cells and incubated for 15 min at room temperature in the dark. The samples were imaged using a fluorescent microscope (Nikon Eclipse E600) with 485/535 nm (FITC) filter and images were analyzed by ImageJ (1.0).
For trypan blue staining, 10 μl 0.4% solution of trypan blue was mixed with 10 μl of cells at 5E5 cells/ml. The mixture was counted on a hemacytometer immediately after mixture.
For PI staining, 1E6 D. discoideum cells were treated with 1 μM TSA, 5 μM sinefungin, or 10 μM BIX 01294 for 6 h. Cells were subsequently washed and resuspended with cold PBS supplemented with 5% FBS and stained with 2 mg/ml propidium iodide (BD Biosciences) at room temperature for 10 min. Stained cells were fixed with 1% PFA. Samples were analyzed by BD LSR Fortessa (BD Biosciences) and data analysis was performed using Flow Jo (Tree Star).

Wang S.Y., Pollina E.A., Wang I.H., Pino L.K., Bushnell H.L., Takashima K., Fritsche C., Sabin G., Garcia B.A., Greer P.L, & Greer E.L. (2021). Role of epigenetics in unicellular to multicellular transition in Dictyostelium. Genome Biology, 22, 134.

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Endoplasmic Reticulum Stress Response

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17-AAG (AAJ66960-EX3), AUY-922 (101756–820),17-DMAG (102513–662), RIPA buffer (AAJ63306-AP), Trypan Blue (76180–676), sheep anti-mouse IgG HRP-linked polyclonal antibody (95017–554), donkey anti-rabbit IgG HRP-linked polyclonal antibody (95017–556), EZBlock™ protease inhibitor cocktail (75837–938) and nitrocellulose membranes (10063–173) were obtained from VWR (Radnor, PA). The ATF-6 (65,880 s), PERK (5683 s), Phospho-PERK (Thr980) (3179 s), IRE-1α (3294 s), BiP (3183 s), CHOP (5554 s), PDI (2446 s), and Ero1-Lα (3264 s) antibodies were purchased from Cell Signaling (Danvers, MA). The β-actin (A5441–0.5 ml) antibody was purchased from Sigma-Aldrich (St Louis, MO). The phospho-IRE1α antibody (Ser724) (16927) was obtained from Thermo Fisher Scientific (Waltham, MA).

Uddin M.A., Kubra K.T., Sonju J.J., Akhter M.S., Seetharama J, & Barabutis N. (2020). Effects of Heat Shock Protein 90 Inhibition In the Lungs. Medicine in drug discovery, 6, 100046.

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Microglia Uptake of Monomeric and Fibrillar α-Synuclein

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Rat-derived primary microglia were pre-incubated with scavenger receptor-targeting treatments, including either 10 µg/mL SRA1 (R&D) and CD36 (Abcam) antibodies or NPs for 24 hours, followed by 24 hour co-incubation with 5 µM monomeric ASYN (rPeptide), ^{48 (link)} fluorescently labeled with DyLight 594 nm (Dy594) microscale antibody labeling kit (Thermo Fischer) or 5 µM monomer-equivalent fibrillar ASYN. For fibrillar ASYN studies, cells were fixed prior to characterization by immunocytochemistry. For monomeric ASYN studies using live cells, extracellular non-internalized synuclein fluorescence quenched by adding 0.5 mg/mL trypan blue (VWR) for 30 minutes, followed by two washes with PBS ^{49 (link)}. Live cells were imaged on a Leica SP2 confocal microscope using a 40 x dry objective. Intracellular fluorescence quantification was performed using NIH-ImageJ software (http://rsb.info.nih.gov/ij/), by measuring mean grey value in cells segmented by applying the same fluorescence thresholds to all collected images. For untreated cell controls, fluorescence images were segmented based on cell boundaries visualized in brightfield images.

Bennett N.K., Chmielowski R., Abdelhamid D.S., Faig J.J., Francis N., Baum J., Pang Z.P., Uhrich K.E, & Moghe P.V. (2016). Polymer Brain-Nanotherapeutics for Multipronged Inhibition of Microglial α-Synuclein Aggregation, Activation, and Neurotoxicity. Biomaterials, 111, 179-189.

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Biotinylation of HIBCPP Cells on Transwell Inserts

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For all biotinylation experiments, cells were grown on Transwell inserts (24 mm diameter, 0.4 µm pore size, polystyrene) (Corning Inc.). HIBCPP cells were lifted with 0.05% trypsin or Accutase for 15 min at 37 °C, followed by manual dissociation with a 1 mL pipette. Growth media was added to stop trypsinization or Accutase digestion, and viable cells were counted using Trypan blue (VWR, Radnor, PA, USA). Then, 1.2 × 10⁶ cells were added to the upper chamber of a 6-well Transwell insert and media was added so that the total volume was 1.6 mL in the upper chamber only. The day after seeding, non-adhered cells were removed and growth media was changed in the upper chamber, and 2.5 mL of growth media was added to the lower compartment. Every 2–3 days thereafter, media was aspirated, cells were washed once with fresh media, and then fresh media was added to the upper and lower chambers of each well. Cells were used for biotinylation experiments when they reached confluence, which was typically 7 days after seeding.

Morgan S.E., Schroten H., Ishikawa H, & Zhao N. (2020). Localization of ZIP14 and ZIP8 in HIBCPP Cells. Brain Sciences, 10(8), 534.

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Spleen Dissection and Cell Isolation

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During dissection, spleens were removed and collected in phosphate buffered saline (PBS) + 2% fetal calf serum (FCS, Gibco) at 4°C. Single cells were obtained after mashing the spleen through a 70 µm nylon cell strainer (VWR) followed by treatment with a RBC lysis buffer (0.83% ammonium chloride (NH₄Cl; Acros Organics, Geel, Belgium)/10 mM Tris (Sigma) solution with pH 7.2) at 37°C. After washing with PBS + 2% FCS, cells were resuspended in PBS + 2% FCS and live leukocytes were counted in a Bürker chamber after 1/2 dilution in trypan blue (VWR).

Pollenus E., Pham T.T., Vandermosten L., Robalo Q., Possemiers H., Knoops S., Opdenakker G, & Van den Steen P.E. (2021). CCR2 Is Dispensable for Disease Resolution but Required for the Restoration of Leukocyte Homeostasis Upon Experimental Malaria-Associated Acute Respiratory Distress Syndrome. Frontiers in Immunology, 11, 628643.

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Inducing Stress Responses in Entamoeba histolytica

Cited 2 times

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To induce ER stress, log-phase trophozoites were incubated with either Dithiothreitol (DTT; Sigma-Aldrich St. Louis, MO, USA) or Brefeldin A (BFA; Thermo Scientific; Hercules, CA, USA) (Delgado-Corona et al., 2002 (link); Manning-Cela et al., 2003 (link); Oslowski and Urano, 2013 (link)) in 13 mL of TYI-S-33 culture medium for 1 h at 37°C. Control cells were incubated with appropriate diluents, sterile water or dimethyl sulfoxide (DMSO; MP Biomedicals, Solon, OH, USA) for DTT and BFA, respectively. To induce nitrosative stress, log-phase trophozoites were incubated with either sodium nitroprusside (SNP; Sigma-Aldrich) (Santi-Rocca et al., 2012 (link)) or dipropylenetriamine NONOate (DPTA NONOate; Enzo Life Sciences, Farmingdale, NY, USA) (Vicente et al., 2009 (link)) in 13 mL of TYI-S-33 culture medium for 1 h at 37°C. After each treatment, trophozoites were incubated on ice for 8 min after stress induction to detach the cells from the glass culture tube. Viability was immediately assessed using microscopy and Trypan Blue (VWR, Radnor, PA, USA) exclusion.

Walters H.A., Welter B.H., Sullivan WJ J.r, & Temesvari L.A. (2019). Phosphorylation of Eukaryotic Initiation Factor 2-α in Response to Endoplasmic Reticulum and Nitrosative Stress in the human protozoan parasite, Entamoeba histolytica. Molecular and biochemical parasitology, 234, 111223.

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MC3T3-E1 Cell Culture and Characterization

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Bovine serum albumin (BSA), fibrinogen from human plasma (FBG), GRGDSP peptide, collagen type I from rat tail, acetic acid, eriochrome black T, phosphate buffered saline (PBS), and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis, MO). Alpha-minimum essential medium (α-MEM) with nucleosides, calcium chloride dihydrate (CaCl₂●2H₂0), fetal bovine serum (FBS), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), and a live/dead cytotoxicity kit for mammalian cells were purchased from Thermo Fisher Scientific (Hampton, NH). Penicillin-streptomycin, tris hydrochloride, trypan blue, trypsin (0.25%) EDTA (1x), trypsin soybean inhibitor, paraformaldehyde, hematoxylin, and potassium chloride (KCl) were purchased from VWR (Radnor, PA). BTT 3033 (1-(4-Fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide) was purchased from R&D Systems (Minneapolis, MN). MC3T3-E1 subclone 14 cells (batch number 61723894) were purchased from the American Type Culture Collection (ATCC; CRL-2594) (Manassas, VA).

Haag S.L., Schiele N.R, & Bernards M.T. (2021). Enhancement and Mechanisms of MC3T3-E1 Osteoblast-like Cell Adhesion to Albumin through Calcium Exposure. Biotechnology and applied biochemistry, 69(2), 492-502.

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LPS-Induced Inflammatory Response Study

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Bacterial lipopolysaccharide (LPS; Escherichia coli O55:B5) was obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). RPMI 1640 cell culture medium and FBS were from VWR International (West Chester, PA, USA). Penicillin and streptomycin were from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The poly (lactide-co-glycolide) copolymer [PLGA; lactide:glycolide (50:50); MW=30,000–60,000] was from Sigma-Aldrich; Merck Millipore. Trypan blue was from VWR International.

Shao P., Ma L., Ren Y, & Liu H. (2017). Modulation of the immune response in rheumatoid arthritis with strategically released rapamycin. Molecular Medicine Reports, 16(4), 5257-5262.

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Endoplasmic Reticulum Stress Response

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Uddin M.A., Kubra K.T., Sonju J.J., Akhter M.S., Seetharama J, & Barabutis N. (2020). Effects of Heat Shock Protein 90 Inhibition In the Lungs. Medicine in drug discovery, 6, 100046.

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