Erastin (Selleck, Houston, TX, USA), RSL3 (Cayman Chemicals, Ann Arbor, MI, USA), ML162 (Cayman Chemicals), dimethyl fumarate (DMF; Santa Cruz Biotechnology, Dallas, TX, USA), Tert‐butyl Hydroperoxide (TBHP; Santa Cruz), 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB; Santa Cruz), crystal violet (Santa Cruz), monosodium glutamate (TCI, Portland, OR, USA), reduced glutathione (GSH; Millipore‐Sigma, St. Louis, MO, USA) and deferoxamine (DFO, Millipore‐Sigma) were purchased from the indicated companies.
Crystal violet
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Crystal violet is a synthetic dye commonly used in laboratory settings. It functions as a stain, enabling the visualization and identification of cellular structures and microorganisms in microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Agarose, by Thermo Fisher Scientific (3 mentions)
Formaldehyde, by Merck Group (2 mentions)
Szh10 stereo microscope, by Olympus (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ml162, by Cayman Chemical (1 mentions)
Erastin, by Selleck Chemicals (1 mentions)
Reduced glutathione gsh, by Merck Group (1 mentions)
Monosodium glutamate, by Merck Group (1 mentions)
Deferoxamine dfo, by Merck Group (1 mentions)
Transwell migration chamber, by Corning (1 mentions)
Invasion chamber, by Corning (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Methanol, by Santa Cruz Biotechnology (1 mentions)
Zen 2, by Zeiss (1 mentions)
Multiskan fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Transwell chamber, by Corning (1 mentions)
Floid cell imaging station, by Thermo Fisher Scientific (1 mentions)
Modified 24 well boyden chambers, by Corning (1 mentions)
Lcx100 imaging system, by Olympus (1 mentions)
19 protocols using crystal violet
1
Reagent Procurement for Cell Metabolism
2
Soft Agar Colony Formation Assay
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The experiment was performed as previously described, with slight modifications [80 (link)]. Briefly, the base agarose layer was prepared in 6-well culture plates by pouring 2 ml of base agarose mixture comprised of 1X DMEM, 10% FBS, and 0.5% agarose (Affymetrix, cat. #32802). Cells were prepared in top agarose mixture comprised of 1X DMEM, 10% FBS, and 0.35% agarose, and poured over the solidified base agarose layer at final seeding density of 5,000 cells/well. After solidification of the top layer, 2 ml of growth media with 0 or 2 μg/ml DOX were added to each well, and samples were placed in a 37°C incubator. Cells were allowed to incubate for 4 weeks, with the overlaid media and DOX exchanged thrice weekly. At the termination of the experiment, samples were rinsed twice with DPBS and fixed with 3.7% formaldehyde (Sigma-Aldrich, cat. #252549) for 10 minutes. After two more washes with DPBS, cells were stained with 0.01% crystal violet (Santa Cruz Biotechnology, cat. #sc-207460) for 1 hour. Photographs were taken using EVOS FL Imaging System, and colonies were counted using ImageJ (National Institutes of Health, Bethesda, MD). To exclude background, only colonies > 10,000 μm2 were counted.
3
Long-term Osteoblast Cell Culturing
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OSC cells (800 cells/plate) were put into 6 well plates after 48 h transfection, and incubated in medium for 21 days, and then the plates were stained with 0.5% crystal violet (Santa Cruz, Dallas, TX, USA).
4
Colony Forming Efficiency of DBMSCs
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Colony forming efficiency of DBMSCs was assessed as previously published [16 (link)]. Briefly, DBMSCs were seeded into six-well plates at a density of 100 cells/well in complete DBMSC culture medium. The medium was then replaced with fresh medium every 3 days. After 14 days culture as described above, the medium was removed and the cells were then washed with PBS for three times. Cells were then fixed with 4% paraformaldehyde in PBS, pH 7.4 for 30 minutes at RT. After washing cells twice with PBS, they were stained with 0.1% crystal violet (Santa Cruz, Saudi Arabia) for 15 minutes at RT, rinsed with distilled water, and visualized and photomicrographs were recorded as described above. Colonies of cell aggregates of ≥50 cells were scored. Each experiment was performed in triplicate using DBMSCs from passages three to five, from twenty individual placentae.
5
Clonogenic Potential Evaluation of Cancer Cells
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The ability of the cells to form clones was assessed using a clonogenicity test. PC3 luc and PC3–M8 luc cells were cultured in a 6–well plate at a confluence of 800 cells/well for 2 weeks. The medium, which contained different treatment conditions (WS12 at 1 and 10 nM, empty LNCs, WS12–loaded LNCs at 1 and 10 nM, icilin at 10 µM, and M8B at 1 µM), was changed every three days during the time of the experiment. After 2 weeks of incubation, the clones obtained were rinsed with PBS, fixed with methanol, and stained with Crystal violet (Santa Cruz, CA, USA). The number of clones present in each well was counted using ImageJ software [51 ], and each condition was normalized to the control condition.
6
Transwell Migration and Invasion Assay
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Transwell assay was performed using transwell migration chambers (#3422, Costar, Cambridge, MA, USA) or invasion chambers (#354480, Costar). Briefly, ESCC cells re-suspended in cell medium (200 μL) were inoculated into apical chambers after transfection for 24 h. Also, 600 μL medium containing 10% FBS (Thermo Fisher Scientific) was added to basolateral chambers. After culture for a while (24 h), the migrating and invasive cells were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) and stained with 0.1% crystal violet (Santa Cruz Biotechnology). The cells in 5 random fields were figured using a Nikon microscope (× 100, Nikon Eclipse E600, Nikon Instruments).
7
Cell Viability Assay with THBS1/2
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A total of 103 cells/well were plated in 6-well dishes, exposed to treatments, and incubated at 37 °C. One week later, cell colonies were fixed, stained with methanol 25% and crystal violet 0.1% (Santa Cruz Biotechnology, Dallas, TX, USA, sc-207460), and counted using the ZEN 2.0 software (Carl Zeiss, Oberkochen, Germany). Trypan blue stain and the hemocytometer were used to determine viable cell numbers. Cell viability was also measured using the colorimetric 3-(4,5)dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (CyQUANT™ MTT Cell Viability Assay V13154, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cells were grown in 96-well plates and incubated with 1000 ng/mL of rhTHBS1 and rhTHBS2 for 24 h, 48 h, and 72 h. Control cells were prepared in plates containing only medium. At the end of the incubation period, MTT was added to each well and incubation was carried out for 4 h at 37 °C. Living cells reduced MTT to formazan, which was quantified by measuring the absorbance at 570 nm (Multiskan™ FC Microplate Photometer, Thermo Fisher Scientific, Waltham, MA, USA).
8
Cell Proliferation and Colony Assay
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The cells were seeded into petri dishes with a density of 500 cells per dish and incubated for 2 weeks. The cell culture medium was refreshed every 3 days. After 2 weeks, the cells were stained with crystal violet (0.5 mg/mL, Santa Cruz Biotechnology Inc.) for 30 min on a shaker after washing with PBS. After 30 min, the cells were washed with PBS for several times. The colonies were photographed and cell colonies with >50 cells or more were counted.
9
Transwell Assay for Cell Motility
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The motility capabilities of the cells in vitro were measured using Transwell chambers (Corning, Corning Incorporated, Corning, NY, USA). Subsequently, four groups of cells (5×105) were seeded on the upper wells with serum-free medium. Medium with 20% FBS was plated in the bottom wells as chemoattractants. After 48 h incubation, the cells were fixed with 90% methanol and stained with 1% crystal violet (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 30 min at 37°C. Cells staying on the upper side of the membranes were wiped, while those on the lower side were counted and photographed with microscope.
10
Anchorage-Independent Cell Growth Assay
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The base agarose layer was prepared in 6-well culture plates by pouring 2 ml of base agarose mixture comprised of 1X DMEM, 10% FBS, and 0.5% agarose (Affymetrix). Cells were then prepared in the top agarose mixture comprised of 1X DMEM, 10% FBS, and 0.35% agarose, and poured over the solidified base agarose layer at a final seeding density of 5,000 cells/well. After solidification of the top layer, 2 ml of media were added to each well, and samples were placed in a 37°C incubator. Cells were incubated for 4 weeks, and the overlying media changed thrice weekly. At the end of the experiment, samples were rinsed twice with DPBS and fixed with 3.7% formaldehyde (Sigma-Aldrich) for 10 minutes. After two more washes with PBS, cells were stained with 0.01% crystal violet (Santa Cruz Biotechnology) for 1 hour. Pictures were taken using an Olympus SZH10 Stereo microscope (Olympus, Japan), and colonies were counted using ImageJ (http://imagej.nih.gov/ij/ ).
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