Mouse monoclonal antibodies to p21 (sc-187), p53 (DO-1) and β-actin (C4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A mouse monoclonal antibody to BrdUrd (clone BU-33) was purchased from Sigma (St. Louis, MO, USA). An Alexa Fluor 488 secondary antibody (goat anti-mouse IgG) was purchased from Invitrogen (Eugene, OR, USA). The fluorescent tracers 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen), Hoechst 33258 (Invitrogen), propidium iodide (PI) (Sigma), the vital dye trypan blue (TB) (Sigma), and the tetrazolium dyes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) (Roche Diagnostics, Penzberg, Germany) were used as recommended by the manufacturers. Dichloroacetate (DCA) and sodium salicylate (NaSal) (both from Sigma) were dissolved in distilled water and stored at 4 °C.
Trypan blue tb
Manufactured by Merck Group
Sourced in United States
Trypan Blue (TB) is a dye commonly used in laboratory settings for cell viability and counting. It is a blue-colored azo dye that can selectively stain dead cells, allowing for the differentiation between viable and non-viable cells under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Dichloroacetate dca, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyl tetrazolium bromide mtt, by Roche (1 mentions)
Sodium salicylate nasal, by Merck Group (1 mentions)
β actin c4, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
William s e medium, by Thermo Fisher Scientific (1 mentions)
7 ethoxycoumarin, by Merck Group (1 mentions)
Victor x5 multilabel plate reader, by PerkinElmer (1 mentions)
Sulforhodamine b srb, by Merck Group (1 mentions)
Prism software, by GraphPad (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Mc540, by Merck Group (1 mentions)
Ethylene glycol, by Sinopharm (1 mentions)
Nitric acid, by Sinopharm (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
8 protocols using trypan blue tb
1
Antibody Staining and Viability Assays
2
Bioreactor Comparison for Cell Culture
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William’s E medium was purchased from Life Technologies (Grand Island, NY, USA). Trypan blue (TB) and 7-ethoxycoumarin were purchased from Sigma-Aldrich (St. Louis, MO, USA). RB bioreactors were purchased from RealBio® Technology, Inc. (Kalamazoo, MI, USA). Quasi-Vivo® bioreactors were provided by Kirkstall Ltd. (Rotherham, UK). The FB bioreactor was manufactured by Professor Jeffrey M. Macdonald and Vaibhav Hans, University of North Carolina (Chapel Hill, NC, USA).
3
Cell Viability Assays for Drug Screening
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Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide), Sulforhodamine B (SRB, Sigma-Aldrich, Saint Louis, MO, USA) or Trypan Blue (TB, Sigma-Aldrich, Saint Louis, MO, USA). Conversion of MTT by mitochondrial succinate dehydrogenase was used as an indicator of cell viability as described [80 (link)]. The SRB assay, based on cellular protein content, was performed as described [81 (link)]. For TB assay, live and dead cells were counted with an optical microscope after staining. For MTT and SRB assay, plates reading was performed on a Multilabel Plate Reader (Victor X5, PerkinElmer, Waltham, MA, USA) at 570 nm. EC50 was calculated after 24 h as the concentration giving a 50% decrease in the cell number compared to untreated cells using the GraphPad Prism software (Version 6.0, GraphPad, San Diego, CA, USA).
4
Quantifying Microparticle Internalization in Cells
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MCF-10A and SKBR-3 cells were seeded in 35 mm petri dishes at a density of 1.5 × 105 cells/dish. After 48 h, the medium was removed and cells were incubated with Alexa488-IgG-microparticles either non-coated, or coated with PEI-25 K or PEI-750 K at a 5:1 ratio (microparticle:cell), in serum-free medium for 4 h in standard conditions. Microparticle internalization was evaluated at 4 h, when cells were harvested by trypsinization and analyzed under a flow cytometer. In order to distinguish internalized microparticles from those attached to the cell membrane, 2 mg/ml Trypan Blue (TB, Sigma) was used to quench the extracellular fluorescence13 (link)21 (link). Cells were analyzed under a Becton Dickinson FACSCanto II flow cytometer both prior and after TB addition. This method allowed obtaining integrated information about cell viability and microparticle internalization efficiency (% of cells with internalized microparticles). For each PEI treatment and time of incubation, three independent experiments were performed analyzing 20.000 cells each.
5
Synthesis and Cell Culture Evaluation
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All reagents used were of analytical quality. For the synthesis, the following reagents were used: Yb2O3; Er2O3 and Dy2O3 (analytical reagent); nitric acid (HNO3, 65.0%–68.0%); ammonium phosphate monobasic (NH4)2HPO4 (analytical reagent); ethylene glycol; and glycero (99.0%) from Sinopharm Chemical Reagent Co., Ltd., China (Mainland). The water used in this study was deionized by a Milli-Q Plus system, whose electrical resistance was 18.2 MΩ.
For cell culture experiments, the following reagents were used: Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) from HyClone, Thermo Scientific, Logan, Utah, USA; methyl thiazolyl tetrazolium (MTT); trypan blue (TB); dimethyl sulfoxide; MC540; and DOX from Sigma-Aldrich Co., (St Louis, MO, USA). The medium was supplemented by antibiotics to inhibit bacterial growth. Penicillin and streptomycin were used in combination. The final concentration of the penicillin or streptomycin was 100 units per mL. When used, 1 mL glutamine solution (0.2 M) was further added to 100 mL culture medium.
For cell culture experiments, the following reagents were used: Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) from HyClone, Thermo Scientific, Logan, Utah, USA; methyl thiazolyl tetrazolium (MTT); trypan blue (TB); dimethyl sulfoxide; MC540; and DOX from Sigma-Aldrich Co., (St Louis, MO, USA). The medium was supplemented by antibiotics to inhibit bacterial growth. Penicillin and streptomycin were used in combination. The final concentration of the penicillin or streptomycin was 100 units per mL. When used, 1 mL glutamine solution (0.2 M) was further added to 100 mL culture medium.
6
Fluorescent Bacterial Staining Protocols
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The bacterial staining dyes Trypan Blue (TB; Sigma-Aldrich, Tokyo, Japan), SYTO 9 (Thermo Fisher Scientific, Tokyo, Japan), and FM 4–64 (Thermo Fisher Scientific, Tokyo, Japan) were used in this work. Prior to TB staining, 0.4% TB was diluted to 0.1%. (KFF)3K-FAM permeated cells prepared as described above were washed three times with PBS, pelleted and resuspended in 50 μl 0.1% TB. For analysis of TB stained cells with flow cytometry, stained cells were washed once with PBS to remove excessive TB. FM 4–64 and SYTO 9 staining was performed based on the manufacturer’s protocol. Observation of both TB and FM4–64 stained cells with fluorescence microscopy was performed immediately upon resuspension. For SYTO 9 stained cells, cells were washed three times with PBS prior to microscopic observation.
7
Efficient Delivery of Fluorescent Proteins via Electroporation
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The electroporation was realized with two kinds of molecules, solubilized in B1 buffer:
Trypan Blue (TB, Sigma-Aldrich; 0.08% in 70 μl of B1), a dark blue vital stain that enters a cell only if the plasmatic membrane is porated or damaged. It was used when applying protocol P1, to verify the reliability and selectivity of electroporation with capacitive microelectrodes. Before applying the stimulus, TB was incubated 1 min on the cell culture to detect any damaged cell. Twenty minutes after the electroporation, the TB solution was removed, the cells rinsed thrice with PBS and the result checked at the microscope;
pcDNA3.1(-)EYFP/ECFP (2 μg in in 70 μl of B1), with cloned Enhanced Yellow Fluorescent Protein (EYFP) or Enhanced Cyan Fluorescent Protein (ECFP) gene49 (link). Plasmids were delivered to the cells by means of protocol P2. After the electroporation, the plasmid solution was incubated twenty minutes on the cells, then the culture was rinsed thrice with PBS and put in the incubator with the appropriate sterile complete medium. The expression of the fluorescent protein EYFP or ECFP was verified 24 or 48 h after the electroporation.
8
Pharmaceutical Compound Analysis Protocol
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GEST (99%) was provided by Zizhu Pharmaceutical Co., Ltd. (Beijing, China). GEST standard (99.4%) and norgestrel (99.4%, internal standard, IS) were purchased from National Institutes for Food and Drug Control (Beijing, China). Polyvinylpyrrolidone (PVP K-90 and PVPK-30) was obtained from Boai NKY Pharmaceutical Ltd (Beijing, China). CMC Na and chondroitin sulfate were obtained from Baichuan biology technology Ltd (Xian, China). Methanol and acetonitrile were of HPLC grade (Fisher Scientific, Geel, Belgium). Trypan blue (TB) was obtained from Sigma-Aldrich (USA). All other chemicals were of analytical reagent grade.
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