Recovery and viability of MNCs after thawing were assessed using the LUNA Automated Cell Counter (Logos Biosystems, Anyang, South Korea) together with 0.4% Trypan Blue Solution (Lonza, Basel, Switzerland). The cell suspensions were adjusted to 2 × 107 viable cells/mL, and the concentration was verified using a XN-350 Hematology Analyzer (Sysmex, Vienna, Austria).
Trypan blue solution
Manufactured by Lonza
Sourced in Switzerland, United States, Sweden
Trypan blue solution is a dye commonly used in cell biology for determining cell viability. It is a blue dye that selectively stains dead cells, allowing them to be differentiated from live cells under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Sorp 6 laser cell sorter, by BD (2 mentions)
Amaxa nucleofector kit, by Lonza (2 mentions)
Tc20 cell counter, by Bio-Rad (2 mentions)
Mycoalert kit, by Lonza (2 mentions)
Xn 350 hematology analyzer, by Sysmex (1 mentions)
Luna automated cell counter, by Logos Biosystems (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Neurobasal media, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Pge2 d4, by Cayman Chemical (1 mentions)
Milli q system, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
Glutamax 100, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Leibovitz s l 15 medium, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Cis 5 8 11 14 17 eicosapentaenoic acid epa, by Merck Group (1 mentions)
6 keto pgf1α, by Cayman Chemical (1 mentions)
8 protocols using trypan blue solution
1
Cell Viability Assessment after Thawing
2
Quantify Cell Death and Cell Cycle Analysis
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To quantify cell death, 50,000 cells were plated in 12 well plates in triplicate and then either left untreated or treated with different concentration of LzCD95L for 24 hr. The total cell pellet consisting of live and dead cells was either re-suspended in lysis buffer (0.1% sodium citrate, pH 7.4, 0.05% Triton X-100, 50 μg/ml propidium iodide), and after incubating for 2-4 hours in the dark at 4°C, percent cell death was quantified by flow cytometry or cells were re-suspended in media and an equal volume of Trypan blue solution (Lonza) was added. Both living and dead (blue) cells were counted on a hemocytometer under a light microscope. To perform cell cycle analysis, 600,000 cells were plated in 6 well plates in triplicate. After 16-20 hours (at ∼80% confluence), plates were gently rinsed with PBS and trypsinized to obtain cell pellets. Cell pellets were then washed (resuspended in 2.5 ml PBS, centrifuged at 500 x g for 5 minutes, and then decanted) and resuspended in 500 μl lysis buffer (see above) and kept on ice and protected from light. Immediately before FACS analysis, samples were spiked with 1:500 DAPI solution as a counterstain to assess cell viability (with corresponding no DAPI controls). Samples were then analyzed on a BD FacsAria SORP 6-Laser Cell sorter at the RHLCCC Flow Cytometry Core Facility. Subsequent data were analyzed using FlowJo 10 software.
3
Measuring 5hmdC and 5fdC in cells
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Cell lines were routinely tested for mycoplasma contamination using Lonza Mycoalert Kit. Cell proliferation assays were done by seeding cells in p60 plates or in a T25 flask with appropriate concentrations of 5hmdC, 5fdC or dC in the growth media. The cells were passaged, counted and the media was replaced every two days. Before counting, 1 volume of Trypan blue solution (Lonza) was added to an aliquot of single cell suspension. The live cells were counted by TC-20 Cell Counter (Bio-Rad). NTPs were introduced by nucleofection. 106 MDA-MB-231 cells were nucleofected with 50 mM 5hmdC in a 100 μl volume using an Amaxa nucleofector kit (Lonza), following the manufacturer’s instructions. After transfection, cells were seeded in a 6 well plate, 24 h later washed twice with PBS, and 48 h later DNA extracted for HPLC analysis.
4
Measuring 5hmdC and 5fdC in cells
5
Cell Proliferation Assay for Ruta graveolens
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For in vitro cell proliferation assay, MTT and Trypan blue exclusion tests were performed. MTT assay : cells were seeded at 3x105 cells/well in a 24 well plate in the presence or absence of 1mg/ml of R graveolens a.e., and cell proliferation assessed after 24, 48 and 72 hours. According to manufacturer’s recommendations, 50 μl of 3-(4,5 dymethylthiazol-2-il)-2,5 dyphenyl-2H-tetrazolium bromide (MTT) reagent (5mg/ml in PBS) was added to each well and, then, the cells were incubated at 37°C for three hours. One volume (500μl) of Stop mix solution (20% SDS in 50% dimethyl formamide) was added to each well and incubated at room temperature for a minimum of 1h. The plate was read at 550nm and at 630 nm as the reference wavelength. Same volume of medium without cells was used as blank. Results are expressed as OD.
Trypan blue exclusion test : cells were seeded at 3x105 cells/well in a 24 well plate in presence or not of 1mg/ml of R. graveolens a.e. Cell proliferation was assessed 24, 48 and 72 h after R. graveolens a.e. addition. Cells were harvested and resuspended in 1ml of PBS. 0.2 ml of cell suspension were added to 0.5 ml of PBS and 0.3 ml of 0.4% of Trypan blue solution (Lonza, Walkersville, MD, USA). After 5 min at room temperature, cells were counted in a Burker’s chamber.
6
Isolation of Primary Rat Cortical Neurons
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All procedures followed NIH and federal guidelines and were approved by the University of Pittsburgh, the Institutional Animal Care & Use Committee. Primary neurons were isolated from E18 rat fetus (Taconic). In brief, E18 pregnant rats were euthanized by CO2 and the rat pups extracted. Pup brains were removed, and cortices were dissociated by Trypsin/EDTA solution (0.05%) followed by mechanical trituration with a fire-polished Pasteur pipette. Dissociated cells were collected by centrifuge (200 g, 5 min) and resuspended in NeuroBasal media (Gibco) supplemented with 2% B27 (Gibco) 1% GlutiMax (Gibco) and 1% PenStrep (Life Technologies). Cells were counted by taking a small aliquot of cell suspension and mixing 1:1 in Trypan blue solution (0.4%, BioWhittaker), following which cells were plated at a density of 2.5 × 105 cells/cm2.
7
Prostaglandin and Prostacyclin Quantification
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Prostaglandins PGE2 (99%) and PGE3 (98%); prostacyclins 6-keto-PGF1α (98%) and Δ17-6-keto-PGF1α (98%); deuterated internal standards PGE2-d4 (99%) and 6-keto-PGF1α-d4 (99%) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Acetonitrile (99.8%), methanol (99.8%) and formic acid (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A Millipore Milli-Q system was used to produce ultra-pure water 18 MΩ (Millipore, Milford, MA, USA). Cis-5,8,11,14-eicosatetraenoic acid (ARA, 85%) and cis-5,8,11,14,17-eicosapentaenoic acid (EPA, 99%), were purchased from Sigma–Aldrich (Oslo, Norway). Leibovitz`s L-15 medium and laminin (cat#L2020) were from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS, cat# 14-801F) was from BioWhittaker (Petit Rechain, Belgium). The glutaMaxTM 100 × (cat# 35056) and the collagenase type IV (cat#17104019) were from Gibco-BRL (Cergy-Pontoise, France). The penicillin-streptomycin mixture (cat#17-602E) and the trypan blue solution (cat#17-942E) were from Lonza (Falun, Sweden).
8
Quantitative Cell Death and Cycle Analysis
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To quantify cell death, 50,000 cells were plated in 12 well plates in triplicate and then either left untreated or treated with different concentration of LzCD95L for 24 hr. The total cell pellet consisting of live and dead cells was either re-suspended in lysis buffer (0.1% sodium citrate, pH 7.4, 0.05% Triton X-100, 50 μg/ml propidium iodide), and after incubating for 2-4 hours in the dark at 4°C, percent cell death was quantified by flow cytometry or cells were re-suspended in media and an equal volume of Trypan blue solution (Lonza) was added. Both living and dead (blue) cells were counted on a hemocytometer under a light microscope. To perform cell cycle analysis, 600,000 cells were plated in 6 well plates in triplicate. After 16-20 hours (at ~80% confluence), plates were gently rinsed with PBS and trypsinized to obtain cell pellets. Cell pellets were then washed (resuspended in 2.5 ml PBS, centrifuged at 500 x g for 5 minutes, and then decanted) and resuspended in 500 µl lysis buffer (see above) and kept on ice and protected from light. Immediately before FACS analysis, samples were spiked with 1:500 DAPI solution as a counterstain to assess cell viability (with corresponding no DAPI controls). Samples were then analyzed on a BD FacsAria SORP 6-Laser Cell sorter at the RHLCCC Flow Cytometry Core Facility. Subsequent data were analyzed using FlowJo 10 software.
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