IHOKs were plated and cultured to 70% confluency. To evaluate the migratory activity of each cell line, we used each cell line's own culture medium to fill the lower chamber, for the migratory potential was altered by changing the medium. In brief, 2 × 104 cells/well were resuspended either in F‐medium without FBS or in KGM without the supplementary bullet kit. Then, the cells were seeded in the upper wells of 24‐well transwell chambers (Coster, Cambridge, MA) for 48 hr. The cells' own media were placed in the lower wells, and the cells migrated to the lower wells. After 48 hr incubation in 5%CO2 incubator, cells were fixed in 10% formalin solution and stained in 0.025% crystal violet (Junsei, Japan). Then, the number of migratory cells was counted in five fields at 200× magnification. The mean for each chamber was calculated and was normalized by counting the number of IHOK cells that were separately incubated in the same condition.
Crystal violet
Manufactured by Junsei
Sourced in Japan
Crystal violet is a synthetic dye commonly used in laboratory settings. It is a dark purple crystalline solid that is soluble in water and alcohol. Crystal violet serves as a basic stain in various microscopic and microbiological techniques, such as Gram staining, which is used to differentiate between Gram-positive and Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Ras 3000 image analysis system, by Fujifilm (2 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Crystal violet, by Bio-Rad (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Dualed blue white transilluminator, by Bioneer (1 mentions)
7 protocols using crystal violet
1
Transwell Assay for Cell Migration
2
ZIKV Propagation and Titration in Cell Lines
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A549, JAr, and African green monkey kidney epithelial (Vero) cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) were used for this study. A549 and JAr cells were cultured at 37 °C in RPMI 1640 medium (Corning Mediatech, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Corning Mediatech) and 1% antibiotics. Vero cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM; Corning Mediatech) supplemented with 10% FBS and 1% antibiotics. Human neural progenitor cells (hNPCs) were generated as previously described [23 (link)].
ZIKV MR766 (African origin) and PRVABC59 (Asia origin) were purchased from ATCC and propagated in Vero cells. Viral titers were determined using a standard plaque assay [9 (link),23 (link)]. First, viral supernatants were diluted in medium and added to Vero cells seeded in 6-well plates. Viral supernatants were removed after 2 h of attachment and cells were overlaid with DMEM containing the following: 1 mg/mL bovine serum albumin; 40 mM MgCl2; 0.2% glucose; 2 mM sodium pyruvate; 4 mM L-glutamine, 1× pen-strep; and 0.1% NaHCO3. After 7 days of incubation, 10% trichloroacetic acid (Sigma-Aldrich) in PBS was used to fix the infected cells, and 0.5% crystal violet (JUNSEI, Tokyo, Japan) solution was used to stain the cells, in order to quantify the plaque-forming units.
ZIKV MR766 (African origin) and PRVABC59 (Asia origin) were purchased from ATCC and propagated in Vero cells. Viral titers were determined using a standard plaque assay [9 (link),23 (link)]. First, viral supernatants were diluted in medium and added to Vero cells seeded in 6-well plates. Viral supernatants were removed after 2 h of attachment and cells were overlaid with DMEM containing the following: 1 mg/mL bovine serum albumin; 40 mM MgCl2; 0.2% glucose; 2 mM sodium pyruvate; 4 mM L-glutamine, 1× pen-strep; and 0.1% NaHCO3. After 7 days of incubation, 10% trichloroacetic acid (Sigma-Aldrich) in PBS was used to fix the infected cells, and 0.5% crystal violet (JUNSEI, Tokyo, Japan) solution was used to stain the cells, in order to quantify the plaque-forming units.
3
Bufalin Inhibits Colony Formation
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Cells were seeded into 24-well plates (1–2 × 103 cells/well) and treated with the indicated concentrations (0, 20, 40, and 80 nM) of bufalin for 24 h and then washed with PBS to remove bufalin. Thereafter, cells were cultured for the next 7 to 10 days to form colonies. Colonies were stained with Crystal Violet (in 60% methanol; Junsei Chemical Co., Ltd., Tokyo, Japan) and images were acquired using the RAS-3000 Image Analysis System (FujiFilm, Tokyo, Japan). To quantify the numbers of colonies, Crystal Violet dyes were extracted from colonies using 10% acetic acid and the optical density of the resolved Crystal Violet dye was measured at 570 nm.
4
EGCG Modulates Colony Formation
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Cells were seeded in 35 mm culture dishes (2×103 cells/dish) and allowed to grow for 7 to 10 days in the presence of and/or absence of EGCG to form colonies. Colonies of more than 50 cells were visualized by crystal violet (in 60% methanol, Junsei Chemical, Japan) staining and images were taken by RAS 3000 Image Analysis System (Fuji Film, Japan).
5
Clonogenic Assay for PFHxS Toxicity
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Cells seeded into 24-well plates (4 × 104 cells/well) were cultured overnight and then treated with different concentrations (0, 1, 10, 50, 100, 200, 300, 400, and 500 μM) of PFHxS for 12–14 days to allow colony formation. Colonies consisting of more than 50 cells were stained with 0.5% crystal violet (Junsei Chemical Co., Ltd., Tokyo, Japan) in 60% methanol. Images were acquired using the Chemi-Doc XRS imaging system (Bio-Rad, Hercules, CA, USA), and the number of colonies was quantified by measuring the optical density of the extracted crystal violet dye with DMSO at 570 nm.
6
Clonogenic Assay for Cell Survival
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For mock control, Myc-YOD1, Flag-USP21, or both Myc-YOD1 and Flag-USP21-transfected cells (1 × 103), 100-mm dishes were used for seeding. After 14 days, the cells were stained with crystal violet (27210‑0350, Junsei, Tokyo, Japan) to visualize colonies. Culture plates containing colonies were captured using a DUALED Blue/White Transilluminator (A-6020, Bioneer, Daejeon, Korea), and images were obtained. The number of colonies was counted using Image J (National Institutes of Health, Bethesda, MD, USA) after washing with PBS.
7
Transwell-Based Cell Invasion Assay
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An 8 µm pore size Transwell system was coated with Matrigel for 1 h at room temperature. The cells were seeded in 24-well plates at a density of 1 × 105 (PANC-1) and 2 × 105 (Capan-1). siRNA transfection was conducted afterward. The cells then were transferred to the transwell at a density of 0.7 × 105 (PANC-1) and 2.5 × 105 (Capan-1) and incubated for 24 h. The transwell plate was washed with PBS and fixed in 4% paraformaldehyde. The transferred cells were stained with crystal violet (JUNSEI, Tokyo, Japan).
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