Suspensions of 1.0×103 cells were seeded into 96-well plates. After 24, 48, 72 or 96 h of incubation at 37°C, EZ-cytox Cell Viability Assay kit mixture (DoGen, Seoul, Korea) was added. After 1 h of incubation, the fluorescence intensity ratio from 650 to 450 nm was measured. Suspended AGS (1×103) cells were added to each well of a 6-well plate and incubated at 37°C for seven days. Colony forming cells were stained using 0.5% crystal violet staining solution (Sigma-Aldrich; Merck KGaA) and counted. Transwell chambers (Corning Inc., Corning, NY, USA) were coated with fibronectin (Sigma-Aldrich; Merck KGaA). Cells were suspended in serum-free media and seeded into the upper chamber at a density of 1×105 cells (MKN74) and 1.5×104 cells (AGS) per well, and serum-containing media was placed into the lower chamber. After incubation at 37°C for 18–20 h, cells penetrating the pores were stained using 0.5% crystal violet staining solution (Sigma-Aldrich; Merck KGaA) and observed under a light microscope. Suspended MKN74 cells (5×104) were plated into each well of Culture-Insert 2 Well in µ-Dish 35 mm (ibidi Gmbh, Martinsried, Germany). After 24 h of incubation at 37°C, cells were gently removed from the Culture-Insert using sterile tweezers. The used dish was filled with cell-free medium and observed after 24, 48 and 72 h under a light microscope.
Crystal violet staining solution
Manufactured by Merck Group
Sourced in United States, Germany, China
Crystal violet staining solution is a laboratory reagent used in various biological applications. It is a purple-colored dye that stains certain cellular structures, making them visible under a microscope. The solution is typically used in microbiology and histology to stain and visualize cells, tissues, or microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Transwell chamber, by Corning (2 mentions)
Matrigel, by BD (2 mentions)
Elisa reader, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Merck Group (1 mentions)
Culture insert 2 well in dish 35 mm, by Ibidi (1 mentions)
Cell counting kit 8 (cck8), by MedChemExpress (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
24 well upper chamber, by Corning (1 mentions)
Matrigel matrix, by Corning (1 mentions)
Transwell insert, by Corning (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Eclipse e200 led microscope, by Nikon (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Optical microscope, by Olympus (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Absorbance reader, by Agilent Technologies (1 mentions)
Cck 8 assay kit, by Dojindo Laboratories (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
49 protocols using crystal violet staining solution
1
Gastric Cancer Cell Proliferation and Migration Analysis
2
Colony Formation Assay Protocol
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Cells in both control and experimental groups were seeded in six-well plates at 500 cells/well in triplicate and maintained under standard conditions for 10–14 days. After fixation, the cells were stained with a crystal violet staining solution (Sigma-Aldrich, China). Colonies were counted and imaged under a microscope.
3
Evaluating JEV-LAV Cytotoxicity in Glioma Cell Lines
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GL261 cells were plated in 12-well plates and infected with JEV-LAV at an MOI of 0, 1, 5, and 25. The cells were stained with crystal violet staining solution (Sigma) for 5 min after 48 h of the infection.
GL261 cells were plated in 96-well plates and infected with JEV-LAV at an MOI of 0, 1, 5, and 25. The cell survival rates were calculated by using the Cell Counting Kit-8 (MedChem Express) after 48 h of the infection.
GL261, A172, T98G, U87, U251, 4T1, 3T3, and A549 cells were plated in 96-well plates and infected with JEV-LAV at an MOI 25. Cytotoxicity was evaluated by using CCK8 after 48 h of the infection.
GL261, U87, 4T1, and A549 cells were infected with JEV-LAV at an MOI of 10 for 72 h, and the resultant supernatant was collected. The viral titers in the cell supernatant were quantified by TCID50 with BHK21 cells.
GL261 cells were plated in 96-well plates and infected with JEV-LAV at an MOI of 0, 1, 5, and 25. The cell survival rates were calculated by using the Cell Counting Kit-8 (MedChem Express) after 48 h of the infection.
GL261, A172, T98G, U87, U251, 4T1, 3T3, and A549 cells were plated in 96-well plates and infected with JEV-LAV at an MOI 25. Cytotoxicity was evaluated by using CCK8 after 48 h of the infection.
GL261, U87, 4T1, and A549 cells were infected with JEV-LAV at an MOI of 10 for 72 h, and the resultant supernatant was collected. The viral titers in the cell supernatant were quantified by TCID50 with BHK21 cells.
4
Cell Colony Formation Assay
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5690 cells from pre-treated spheroids and organoids were transferred to 100 mm plates (100 cells/mm2) and incubated for 14 days in cell-appropriate media as previously published in our lab [29 (link)]. After that, plates were washed with PBS and incubated with 0.05% Crystal Violet staining solution (Sigma-Aldrich) for 10 min. Plates were again washed 3 times with PBS and allowed to dry. This process leads to the staining of cellular nuclei with a deep purple color. Once dried, plates were photographed for the presence of cell colonies and analyzed.
5
Characterization of OMWW A009 Extracts
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The OMWW A009 extracts were provided by the “Azienda Agricola-Fattoria La Vialla”, following purification as described below. Four batches from different preparations were characterized for molecular contents [10 (link),15 (link),16 (link)], revealing similar molecules (Supplementary Table S1 , Figure 1 ). The two most recent batches produced, termed L3 and L4 respectively, were used in this study. Hydroxytyrosol (HyT), purity ≥ 98%, was purchased from Sigma Aldrich (Sigma-Aldrich, Saint Luis, MO, USA) and used as a reference oil polyphenol. For treatments, HyT was dissolved in 100% ethanol (EtOH) and diluted to the same concentration present in A009 in RPMI 1640 medium. Medium with EtOH alone at the same dilution was used as control vehicle for HyT. Crystal violet staining solution and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide were purchased from Sigma Aldrich (Milan, Italy).
6
Colony Formation Assay for Melanoma
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The ability of cells to form colonies was evaluated on a monolayer surface. After melanoma cells were transfected with USP4 shRNA lentiviral vector, adherent cells were trypsinized and counted, and viability was determined. Of those viable cells, 2000 cells were reseeded in a 6‐well plate (in triplicate). Cells were allowed to adhere and grow for between 12 and 14 days. To visualize colonies, cells were fixed in 100% ethanol and stained with crystal violet staining solution (Sigma‐Aldrich). Colonies were counted with an Image J software.
7
TRPM8-Mediated Colony Formation Assay
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A colony formation assay was performed as described previously [47 (link), 52 (link)]. Approximately equal amounts of PANC-1 cells transfected with vector, wild-type or mutant TRPM8 were seeded in 12-well plates and allowed to grow for 7~10 days. The medium was replaced every 3 days. Cells were washed twice with PBS, fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet staining solution (Sigma-Aldrich, USA). Colonies with more than 50 cells in triplicate wells were counted.
8
Transwell Assay for HCC Migration and Invasion
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Approximately 1×105 HCC cells were suspended in serum-free medium and added in either top-chambers that were not coated with Matrigel for migration assay or Matrigel-coated chambers with 8 μm pores (3422, Corning Costar) for invasion assay. The culture medium containing 10% FBS was added to the bottom chamber. After incubation at 37 °C for eighteen hours, HCC cells may migrate to the bottom chamber through the membrane. Membranes were washed three times with 1×PBS, fixed in 4% paraformaldehyde, and stained with crystal violet staining solution (1.09218, Sigma). The number of migrated or invaded cells through the membrane was separately counted under the microscope from 10 random fields of view.
9
Quantifying Leukemia Cell Viability
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For flow cytometry analysis, leukemia cells were stained with Propidium iodide (PI) for 15 min at room temperature, washed with FACS buffer and analyzed using a CytoFLEX-S cytometer as described above. For a plate assay, 50,000 CA1a cells were seeded per well in 24-well plates and treated with ASO-electroporated RBCEVs. After 3 days, the cells were washed once with PBS, and incubated with 0.5% crystal violet staining solution (Sigma Aldrich). Plates were then washed three times in a stream of tap water and air dried. Afterwards, 500 µl of 50% acetic acid was added into each well and the optical density was measured at 570 nm using the Biotek micro-plate reader.
10
Invasion Assay for U87 and LN229 Cells
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For invasion assays, U87 and LN229 cells (5 × 104 cells/well resuspended in 200 mL MEM or DMEM) were added to the 24-well upper chambers (8-µm pore size; Corning Co., Corning, USA) with Matrigel (BD Biosciences). Next, 600 µL of DMEM containing 10% FBS was added to the bottom chamber. After 24 h of incubation, the culture medium in the upper chamber was discarded and the non-migrated cells were removed. The cells passed through the membrane were fixed with methanol and stained with a 1% crystal violet staining solution (Sigma-Aldrich). Additionally, five randomly selected fields from various angles were used for cell counting, image capturing, and observation under a 400x magnification microscope.
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