Equal volumes of FN and AN cultures adjusted to OD590 0.1 were mixed and dispensed into 24-well polystyrene tissue culture plates. Before incubation, TM or TQ was added to the culture at a final concentration of 0.1%. After anaerobic incubation at 37°C for 24 h, the amount of biofilm mass formed on the bottom of the wells was assessed using crystal violet (Wako Pure Chemical) staining. After removing the test media, the biofilms were gently washed three times with 1 ml saline and then stained with 0.4 ml crystal violet solution (0.01%) at room temperature for 1 h. Excess crystal violet solution was removed and the stained biofilm was washed with 1 ml saline before eluting the remaining dye with 1 ml acetic acid (33%, Wako Pure Chemical) and gentle agitation for 30 min at room temperature. The eluent was transferred to a 96-well plate for measurement at OD550 or OD600 to compare the biofilm mass.
Crystal violet
Manufactured by Fujifilm
Sourced in Japan
Crystal violet is a synthetic dye commonly used in laboratory settings. It is a purple-colored crystalline solid that serves as a staining agent for various biological and microbiological applications. The core function of crystal violet is to provide a visible contrast for the identification and visualization of cellular structures, tissues, and microorganisms during microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Bz x710, by Keyence (3 mentions)
Formaldehyde, by Fujifilm (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
24 well transwell system with 8.0 m pores, by Corning (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Transwell insert, by Corning (2 mentions)
Glutaraldehyde, by Fujifilm (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Biocoat matrigel invasion chamber, by Corning (2 mentions)
Neutral buffered formalin, by Muto Pure Chemicals (2 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (2 mentions)
Methylcellulose, by Fujifilm (2 mentions)
Acetic acid, by Fujifilm (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by ReproCELL (1 mentions)
Wst 1, by Roche (1 mentions)
65 protocols using crystal violet
1
Biofilm Formation Quantification Assay
2
Crystal Violet Staining of HEEpiCs
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Crystal violet staining was executed as previously described.3 (link)20 (link) First, the media in 6-well plates were removed, and HEEpiCs were washed with D-PBS (−), fixed in 99% methanol, and stained for 5 minutes with 0.5% Crystal violet (Wako Pure Chemical Industries, Osaka, Japan) dissolved in methanol/water. Subsequently, excess staining dye was removed, and the wells were rinsed thoroughly with running water until no additional stain leached from the wells. To determine cell morphological changes, we took photographs in more than 20 fields per well using a 400× objective.
3
Colony-Forming Unit Fibroblast Assay
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The colony-forming unit fibroblast assay was conducted by culturing 2000 sorted cells on a fibronectin-coated 100-mm dish for 14 days in DMEM-GlutaMax supplemented with 20% FBS (Life Technologies), 1% penicillin/streptomycin, and 5 ng/mL of basic fibroblast growth factor (ReproCell), as previously described [35 (link)]. The culture medium was refreshed biweekly. Crystal Violet (1 g) (FUJIFILM Wako Pure Chemical) was diluted in 100 mL of methanol to prepare the Crystal Violet solution. This solution was mixed with an equal volume of 4% paraformaldehyde (PFA). Following filtration, the Crystal Violet/PFA mixture was added to each well, and the dish was incubated for 20 min at 25 °C. Subsequently, cells were rinsed three times with PBS, and colonies were quantified.
4
Cell Viability Measurement Protocols
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Cell viability was measured by crystal violet staining or by a cell proliferation reagent WST-1 (Roche). For crystal violet staining, cells were plated at 4 or 7.5 × 104 in a 12-well plate, cultured for 7 to 14 days, and stained with crystal violet (Wako). For WST-1 assay, U2OS cells were plated at 5 × 103 cells per well in a 96-well plate and cultured for 14 days, followed by the addition of WST-1 reagent to the medium and additional incubation for 20 min. The cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases was measured by absorbance at 450 nm.
5
Fibroblast Migration Assay with hBD-3
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Fibroblasts were seeded in a collagen I-coated 96-well plate at a density of 5×104 cells/well for 6 hours at 37°C. Confluent fibroblast monolayers were then wounded using a wound maker (Essen BioScience, Ann Arbor, MI) to create a uniform cell-free zone in each well and washed with PBS to remove the detached cells. In all experiments, fibroblasts were pretreated with 10 µg/ml mitomycin C for 2 hours before stimulation with hBD-3 for 24-48 hours to exclude the influence of hBD-3-mediated cell proliferation on migration. After stimulation, fibroblasts were stained with 0.5% crystal violet (Fujifilm, Tokyo, Japan), and images were recorded using a phase-contrast microscope (Keyence). Wound area was measured using ImageJ software. In some experiments, cells were pretreated with inhibitors for 2 hours before stimulation with hBD-3.
6
Quantifying Bacterial Biofilm Formation
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Biofilm formation was assayed as described previously (Shao et al., 2019 (link)). Briefly, the bacterial strains were incubated overnight in LB broth and resuspended in fresh LB broth to an OD600 of 0.1. Bacterial suspensions (120 μl) were put into 96-well plates and incubated at 28 °C for 24, 48, 72, and 96 h. The bacterial solutions were discarded and washed three times with distilled water. The biofilm forming bacteria were treated with 150 μl of 0.1% crystal violet (CV; Fujifilm, Tokyo, Japan) for 20 min without shaking. The dye was discarded and washed twice with distilled water. The plate was dried completely, subsequently the biofilm was eluted with 150 μl of 100% ethanol, and the CV were dissolved completely. Finally, the eluted biofilm sample’s absorbance was measured at OD595.
7
Transwell Migration and Invasion Assay
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A 24-well Transwell system with 8.0-µm pores (Corning, Inc.) was used for the migration and invasion assays. Serum-starved cancer cells at a density of 7×104/well were seeded into the upper chamber in a 100 µl volume suspension. After cell attachment, the culture medium was changed to the indicated CM for each group. The final FBS concentration was 5% in the upper chambers and 10% in the lower chambers. After a subsequent period of incubation (36 h for migration assay; 60 h for invasion assay), the upper chambers were fixed with 100% methanol (cat. no. 131-01826; Fujifilm) for 20 min and then stained with 1% crystal violet (cat. no. 031-04852; Fujifilm) for a further 20 min at room temperature, and non-migrated cells were removed using cotton swabs. Subsequently, images were captured, and the number of remaining cells were calculated and quantified in 5 random fields of view under a light microscopic (magnification, ×100) equipped with a digital camera (Olympus Corporation). For the invasion assays, the upper chambers of the Transwell system were coated with Matrigel™ (0.5 mg/ml dilatated in DMEM; Corning, Inc.) overnight at 37°C.
8
Migration Assay of Human PASMCs
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Human PASMCs (5 × 104 cells/well) were seeded on a 24-well Transwell insert with a membrane pore size of 8 μm (#3422, Corning) (23 (link)). Thereafter, they were treated with culture medium containing FBS (1%) in the upper chamber and that containing FBS (10%) and the vehicle (DMSO) or drug in the lower chamber for 24 h. Transwell inserts were fixed in paraformaldehyde (4%) and stained with crystal violet (1%; Fujifilm Wako Pure Chemical). The number of migratory cells was counted from digital images of Transwell inserts using the SMZ1270 stereomicroscope system equipped with a DS-Vi1 color microscope camera and NIS-Elements imaging software (Nikon, Tokyo, Japan).
9
Glucose and AMD3465 Colony Assay
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The cells were seeded in 12-well plates at 5×104 cells/mL with glucose (80 mmol/L) and AMD3465 (2 μM). After incubation for 10 days, the plates were stained with 0.5% crystal violet (Wako, Osaka, Japan) in 4% paraformaldehyde for 5 minutes to count the colony numbers. The cell group containing more than 50 cells was identified as a colony. Each assay was repeated five times.
10
Cell Attachment on Polymer Substrates
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The polymer substrates were immersed in DMEM/F-12 medium containing 10% FBS or in serum-free DMEM/F-12 for 1 h at 37°C prior to cell culture. The cells were seeded onto the polymer substrates at a density of 5 × 104 cells/cm2. The cells were allowed to attach to the substrates in DMEM/F-12 medium containing 10% FBS or in serum-free DMEM/F-12 for the indicated times. The non-attached cells were removed by washing the plates twice with PBS. The attached cells were fixed with 0.1% glutaraldehyde overnight at room temperature. The cells were stained with a 0.2% crystal violet (Wako, Osaka, Japan) solution for 15 min for visualization. After staining, the attached cells in three randomly selected fields were counted using an optical microscope. For the inhibition assay, the cells were treated with 5 mM EDTA for 10 min at 37°C prior to seeding of the cells. The cell attachment assay was performed after EDTA treatment, as described above.
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