HepG2 or HB214 (5 × 105 /well) cells were seeded in a 6-well plate and incubated for 24 h. The Medium was replaced with medium containing treatment in the concentration stated in the different experiments and the respective controls. After incubation for 72 h cells were trypsonized and seeded (1 × 104/well) in a new 6-well plate with fresh medium and allowed to incubate for 14 days. On day 14 wells were washed with PBS and incubated with 6% Glutaraldehyde (#BP2547-1 Fisher) and 0.05% w/v crystal violet (#C581-25 Fisher) for 30 min. After incubation wells were washed with ddH2O and allowed to dry. Plates were then imaged on an Epsom V850 scanner and Image J was used to determine the cell area.
C581 25
Manufactured by Thermo Fisher Scientific
The C581-25 is a laboratory instrument designed for high-throughput DNA and RNA extraction. It utilizes magnetic bead-based technology to provide efficient and consistent nucleic acid isolation from a variety of sample types.
Automatically generated - may contain errors
6 protocols using c581 25
1
Cell Proliferation Assay Using Crystal Violet
2
Plaque Assay for Virus Titer Quantification
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Vero cells were plated in 12-well plates at a density of 2 × 105 per well and incubated for 24 h. Infection supernatants were serially diluted to 10-4 in culture media and overlaid on cells for 1 h. Cells were covered with Eagle’s Minimum Essential Medium (without phenol red, supplemented with 5% FBE, nonessential amino acids, 1 mM sodium pyruvate (VWR, 45000-710, Dixon, CA, USA), 2 mM L-glutamine, 20 U/mL penicillin, and 20 μg/mL streptomycin) with 0.6% agarose (ThermoFisher, 16500100). At 48 hpi, cells were fixed with 10% formaldehyde (FisherSci, F79P-4) for 1 h. Medium was removed, wells were washed with diH2O and stained with a 1% crystal violet (FisherSci, C581-25) and 20% ethanol solution (FisherSci, BP2818-4). Plaque assay datasets are represented as both plaque forming units per milliliter (PFU/mL) and as percentage of virus titer versus the DMSO control.
3
Viral Plaque Assay Protocol
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Vero cells were plated in 12-well plates at a density of 2 × 105 cells/well for 24 h. Viral stocks or infection supernatants were serially diluted to 10−8 in culture media and overlaid on cells for 1 h. Cells were covered with Eagle’s Minimum Essential Medium (without phenol red, supplemented with 5% fetal bovine essence, non-essential amino acids, 1 mM sodium pyruvate (45000-710, VWR), 2 mM L-glutamine, 20 U/mL penicillin, and 20 µg/mL streptomycin) containing 0.6% agarose. 48 h post-infection, cells were fixed with 10% formaldehyde (F79P-4, FisherSci) for 1 h. Medium was removed, wells were washed with diH2O, and cells were stained with a 1% crystal violet (FisherSci, C581-25) in a 20% ethanol solution (BP2818-4, FisherSci). For plaque assay of VEEV TrD nsP3 mutants, virus was serially diluted in virus diluent (phosphate-buffered saline, 1% donor calf serum) and overlaid on BHK-J cells seeded at a density of 1 × 106 cells/well in 6-well plates for 24 h. Plates were stained with neutral red and plaques were counted. All viral entities were titered in triplicate unless otherwise specified.
4
Vero Cell Plaque Assay for Viral Quantification
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Two hundred thousand (2 × 105) Vero cells/well were seeded in a 12-well plate and incubated for 24 h. Viral supernatants were serially diluted in culture media and the dilutions were overlaid on cells for viral attachment for 1 h; 0.6% agarose was diluted with Eagle’s Minimum Essential Medium (without phenol red, supplemented with 5% fetal bovine essence, nonessential amino acids, 1 mM sodium pyruvate (45000-710, VWR), 2 mM l -glutamine, 20 U/mL penicillin, and 20 g/mL streptomycin) in 1:1 ratio. Forty-eight hours post infection, the monolayer was fixed for 1 h with 10% paraformaldehyde (F79P-4, FisherSci). Agar plugs were removed, and cells were stained with 1% crystal violet (FisherSci, C581-25) in a 20% ethanol solution (BP2818-4, FisherSci). Plaques were counted and recorded.
5
Clonogenic Survival Assay after UV-C Exposure
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Cells were plated at single cell density in 6 cm dishes with 275 cells per dish and clonogenic cell survival was assayed similarly to as described previously (5 (link),22–24 (link)). Cells were allowed to adhere overnight following standard culture conditions, washed with 1× DPBS (Thermo Fisher Scientific #14190-144), and exposed to indicated dose of UV-C (254 nm) radiation before the culture medium was replenished and cells were returned to the temperature and CO2-controlled incubator. Fourteen days after exposure, cells were fixed with 4% paraformaldehyde (Fisher Scientific #50-980-487) diluted in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) for 15 min at room temperature. The fixative was removed and cells were washed with 1× PBS and then stained with 0.5% crystal violet (Fisher Scientific #C581-25) diluted in water for 60 min. Excess stain was removed and dishes were washed with water. Dishes were imaged using a E-Gel Imager (Thermo Fisher Scientific). Colonies (defined as a minimum of 50 cells) were counted using FIJI. Percent survival was calculated as the percentage of colonies that grew on the treated dishes relative to the untreated dishes at each indicated dose.
6
Quantitative Colony Formation Assay
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Cells were seeded into 6-well tissue culture plates (5 × 104 to 1 × 105 cells per well, depending on the growth rate) and allowed to adhere overnight in complete cell culture medium. The next day, medium was replaced by complete cell culture medium containing appropriate drugs or DMSO as vehicle control. Cells were exposed to vehicle for 7–10 days or indicated drug for 3–5 weeks, with medium change and fresh drug added twice a week. At the end of treatment, remaining cells were gently washed with PBS, fixed/stained with 0.2% crystal violet (Fisher Scientific #C581-25) in 4% paraformaldehyde and incubated at room temperature for 30 min. Cells were washed three times with water to remove excessive dye and allowed to air dry. Pictures of stained cells were taken using an EPSON Perfection V600 scanner. Colony formation was quantified using the ColonyArea ImageJ plugin which provides information about the intensity percentage taking into consideration not only the area covered by the colonies, but also the intensity of staining as a direct relation to the number of cells in a colony (Guzmán et al., 2014 (link)).
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