For all experiments, cells were counted using trypan blue stain (Lonza Bioscience). For the paraquat proliferation assays, cells were treated with 0.035 M CuSO4 for 36 hr. Following the incubation, cells were treated with 10 mM paraquat (Sigma-Aldrich). Using the trypan blue stain, live and dead cells were counted at indicated time points over a period of 24 hr. For the proliferation assay with paraquat and GSH, cells were treated with 5 mM L-Glutathione reduced (Sigma-Aldrich) for 1 hr before adding CuSO4 inducer. After this 36 hr induction period, 5 mM of L-Glutathione reduced was added again and the cells were incubated for 1 hr before the addition of 10 mM paraquat (Sigma-Aldrich). Cells were then counted at indicated time points over a period of 24 hr using trypan blue stain.
Trypan blue stain
Manufactured by Lonza
Sourced in United States
Trypan blue stain is a laboratory reagent used to distinguish viable from non-viable cells. It is a diazo dye that selectively colors dead cells blue, while living cells remain unstained. This stain is commonly used in cell counting and viability assessment procedures.
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Lab products found in correlation
Paraquat, by Merck Group (2 mentions)
L glutathione reduced, by Merck Group (2 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Hanks balanced salt solution 1x, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 1x, by Corning (1 mentions)
Cd11bhi, by BioLegend (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
Colorview iiiu digital camera, by Olympus (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Ckx41 inverted microscope, by Olympus (1 mentions)
Automated cell counter, by Bio-Rad (1 mentions)
Trypsin, by Lonza (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Lonza (1 mentions)
Valinomycin, by Merck Group (1 mentions)
Succinic acid, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
9 protocols using trypan blue stain
1
Paraquat-Induced Cellular Stress Modulation
2
Cell Viability and Doubling Time Assay
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A total of 2.5 × 103 cells were seeded in quadruplicates in a 96-well plate. Cell viability was measured at 24, 48, 72, and 96 h post-seeding using the CellTilter 96® Aqueous One Solution Cell Proliferation Assay, MTS (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. For the doubling time, cells were trypsinized and manually counted using Trypan Blue Stain (Lonza, Walkersville, MD, USA) at two different time points. Then, cell doubling time was calculated with the formula:
3
In Vivo Mammary Tumor Xenograft Model
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Mouse mammary carcinoma cells were grown to approximately 75% confluence. Balb/C athymic nude mice were anesthetized with isofluorane/O2 and injected bilaterally into the fourth mammary fat pad with 20μl of 5×104 cells/gland or as described otherwise. Cells were suspended in 25% Growth Factor Reduced Matrigel (BD Bioscience, Bedford, MA), containing 10% Trypan Blue Stain (Lonza, Walkersville, MD) and 65% PBS. Tumor growth from both sides were measured twice a week and monitored weekly by in vivo bioluminescent imaging. Tumor volume was calculated using the formula: ½ (LxWxD), where l=length, w = width and d= depth. At 20-30 days after cell injection, mammary primary tumors were removed as a parallel of the human clinical setting. Ten days later, animals were imaged and metastases in lungs were observed over a period of 4-6 weeks. Statistical analysis of differences in tumor volume was performed using Student t-test (GraphPad Software, Inc. 2014 – online version).
4
Human Neutrophil Isolation Protocol
5
Cell Viability Assay on Decellularized Extracellular Matrices
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An amount of 1 × 104 CPCs were seeded on dECM-NH or dECM-PH mounted on 96-well plates and cultured for 7 days under standard culture conditions in the same cell culture medium used for hydration. As a control, 1 × 104 cells were seeded on fibronectin-coated wells. Cells were checked daily by an Olympus CKX41 inverted microscope equipped with a Colorview IIIu digital camera (Olympus Corporation, Tokyo, Japan). Beginning at 48 h after seeding, and then every day for one week, cell death rate was assessed using trypan blue exclusion assay adapting a previously described protocol [58 (link),61 (link)]. Specifically, every day cells were detached from a subset of wells in the multi-well plates by incubation with 0.25% trypsin-EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Detached cells were then stained with trypan blue stain (0.4% in PBS) (Lonza, Walkersville, MD, USA) for 2 min at room temperature and counted using a hemocytometer. The percentage of dead cells and of alive cells over total cells for each time point was expressed as the mean percentages.
6
Cytotoxicity of Substituted Furans in Engineered Cells
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V79 and V79-hCYP2E1-hSULT1A1 cells were exposed to either DMF (0.5, 1.0, 1.5 or 2.0mM) for 0.5h or FFA (1.0, 3.0, 6.0 or 15.0mM) for 3h in flasks (25cm2) and incubated in medium (10ml) at 37ºC. Due to the difference in toxicity between DMF and FFA (Figures 1 and 2), different treatment (concentration and time) were selected. The flasks were sealed tight during exposure due to the volatile nature of the substituted furans. DMF was dissolved in DMSO immediately before exposure; the final concentration of DMSO was ≤0.2%, whereas FFA was dispersed directly into the medium. The cells were washed with Dulbecco’s PBS (pH 7.4) without Ca2+ or Mg2+ prior to adding 1ml of pre-warmed trypsin (170 000U/L)/EDTA (Lonza) and incubated for 7min. trypsination was arrested by adding ice-cold cell medium. Cells were centrifuged (300 × g, 2min) and the pellets were re-suspended in DMEM without FCS and placed on ice. A preparation was made of the cell suspension (20 µl) and Trypan Blue Stain (0.4%, Lonza, Basel, Switzerland) (1:1) to determine cell count and viability using an automated cell counter (BioRad Laboratories, Hercules, CA, USA). The positive assay control consisted of cell samples from each cell line (V79; V79-hCYP2E1-hSULT1A1) being subjected to 225 KeV X-rays filtered through 0.5mm Cu for a total dose of 10 Gy (3.07 Gy/min).
7
Quantifying Microglial Uptake of Amyloid-beta
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FITC-labeled Aβ (FAM-Aβ) oligomers were prepared as previously described [46 (link)]. Treatments with FAM-Aβ oligomers (1 µM), prepared in RPMI 1640 medium without FBS, were added to primary microglial cultures grown in 6-well plates. Different time courses of treatments were explored: 0.5–3 h for internalization and 1–24 h for degradation. After the incubation period, cells were washed with PBS to remove non-internalized FAM-Aβ. Cells were collected by trypsin/EDTA incubation (2.5 mg/ml, 15 min, 37 °C) followed by resuspension with micropipette and centrifugation (400g, 5 min, 4 °C). To discard the signal from FAM-Aβ externally bound to cell membranes, cells were incubated with 0.05% trypan blue stain (Lonza, Italy) in PBS (pH 7.4) for 1 min at RT to quench only extracellular signal, as previously described [62 (link)]. Then, samples were centrifuged (400g, 5 min, 4 °C) to wash out excess trypan blue, and cell pellets were resuspended in FACS buffer and immediately analyzed by flow cytometry [525 BP40 channel (505–545 nm), illumination with a blue laser (488 nm)]. The percentage of FAM-Aβ-positive cells, as well as the FAM-Aβ fluorescence intensity, were analyzed with Kaluza software.
8
Mitochondrial Membrane Potential Assay
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Sucrose, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic
acid (HEPES), potassium hydroxide (KOH), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine
iodide (JC-1), valinomycin, succinic acid, potassium chloride (KCl),
magnesium chloride (MgCl2), and poly(vinyl alcohol) (PVA,
99+% hydrolyzed, 89–98 kDa) were from Sigma-Aldrich (Saint
Louis, MO). Fluorescein was from Molecular Probes (Eugene, OR). Rotenone
was from ICN Biomedicals (Aurora, OH). 5-TAMRA was from AnaSpec (Fremont,
CA). Dibasic potassium phosphate (K2HPO4) was
from Mallinckrodt (Saint Louis, MO). Phosphate-buffered saline (PBS,
10×) was from Bio-Rad (Hercules, CA). Dulbecco’s modified
Eagle medium (DMEM), fetal bovine serum (FBS), and 0.5% trypsin (10×,
5 g/L trypsin, 2 g/L EDTA·4Na, 8.5 g/L NaCl) were from Gibco
(Invitrogen, Carlsbad, CA). Trypan blue stain (0.4%) was from Bio-Whittaker
(Walkersville, MD). Fused-silica capillary tubing (50 μm i.d.,
150 μm o.d.) was from Polymicro (Phoenix, AZ). Additional reagents
and materials are described in theSupporting
Information .
acid (HEPES), potassium hydroxide (KOH), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine
iodide (JC-1), valinomycin, succinic acid, potassium chloride (KCl),
magnesium chloride (MgCl2), and poly(vinyl alcohol) (PVA,
99+% hydrolyzed, 89–98 kDa) were from Sigma-Aldrich (Saint
Louis, MO). Fluorescein was from Molecular Probes (Eugene, OR). Rotenone
was from ICN Biomedicals (Aurora, OH). 5-TAMRA was from AnaSpec (Fremont,
CA). Dibasic potassium phosphate (K2HPO4) was
from Mallinckrodt (Saint Louis, MO). Phosphate-buffered saline (PBS,
10×) was from Bio-Rad (Hercules, CA). Dulbecco’s modified
Eagle medium (DMEM), fetal bovine serum (FBS), and 0.5% trypsin (10×,
5 g/L trypsin, 2 g/L EDTA·4Na, 8.5 g/L NaCl) were from Gibco
(Invitrogen, Carlsbad, CA). Trypan blue stain (0.4%) was from Bio-Whittaker
(Walkersville, MD). Fused-silica capillary tubing (50 μm i.d.,
150 μm o.d.) was from Polymicro (Phoenix, AZ). Additional reagents
and materials are described in the
Information
9
Paraquat-Induced Cell Viability Assay
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For all experiments, cells were counted using trypan blue stain (Lonza Bioscience). For the paraquat proliferation assays, cells were treated with 0.035 M CuSO4 for 36 hr.
Following the incubation, cells were treated with 10 mM paraquat (Sigma-Aldrich). Using the trypan blue stain, live and dead cells were counted at indicated time points over a period of 24 hr. For the proliferation assay with paraquat and GSH, cells were treated with 5 mM L-Glutathione reduced (Sigma-Aldrich) for 1 hr before adding CuSO4 inducer. After this 36 hr induction period, 5 mM of L-Glutathione reduced was added again and the cells were incubated for 1 hr before the addition of 10 mM paraquat (Sigma-Aldrich). Cells were then counted at indicated time points over a period of 24 hr using trypan blue stain.
Following the incubation, cells were treated with 10 mM paraquat (Sigma-Aldrich). Using the trypan blue stain, live and dead cells were counted at indicated time points over a period of 24 hr. For the proliferation assay with paraquat and GSH, cells were treated with 5 mM L-Glutathione reduced (Sigma-Aldrich) for 1 hr before adding CuSO4 inducer. After this 36 hr induction period, 5 mM of L-Glutathione reduced was added again and the cells were incubated for 1 hr before the addition of 10 mM paraquat (Sigma-Aldrich). Cells were then counted at indicated time points over a period of 24 hr using trypan blue stain.
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