Transfections were accomplished using Lipofectamine 2000 (Thermo Fisher, #11668027). Crystal Violet powder was obtained from Sigma-Aldrich (St. Louis, MO; #C0775) and brought into solution with dH2O at 0.1%. Antibodies used in immunocytochemistry are the following: 1) Anti-phospho-Histone H2A.x (Ser139) Antibody, mouse monoclonal, clone JBW301 (EMD Millipore; #05–636) 2) Rabbit anti-53BP1, polyclonal, (Bethyl Laboratories; A300–272A); anti-mouse (Alexa-Fluor 488) and anti-rabbit secondary antibody Cy3 (both from Jackson ImmunoResearch Laboratories; #715–165-150/#711–165-152). ProLong® Gold Antifade agent with DAPI (4′, 6-diamidino-2-phenylindole) was used to mount and counterstain nuclei in immunocytochemistry (Cell Signaling Technology; #8961). Comet assay slides and lysis buffer were acquired from Trevigen (CometSlide™ #4250–200-03; CometAssay® Lysis Buffer #4250–050-01). Low melting agarose (NuSieve® GTG® Agarose; Lonza #50084). SYBR® Gold Nucleic Acid Gel Stain (10,000× concentrate in DMSO) ThermoFisher (Invitrogen™; #S11494) was used to stain comet assay slides.
Crystal violet powder
Manufactured by Merck Group
Sourced in United States, Greece
Crystal violet powder is a laboratory reagent commonly used in various applications. It is a synthetic dye that has a deep purple color. The powder form of crystal violet is used in numerous scientific and medical procedures, but the specific details of its core function are not provided in this unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Methanol, by Merck Group (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Cometassay lysis buffer, by Bio-Techne (1 mentions)
Jbw301, by Merck Group (1 mentions)
Cometslide, by Bio-Techne (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Nusieve gtg agarose, by Lonza (1 mentions)
Ix71 inverted research microscope, by Olympus (1 mentions)
Alp activity assay kit, by Abcam (1 mentions)
5α dht, by Merck Group (1 mentions)
Alpha modified minimal essential medium α mem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Ab16491, by Abcam (1 mentions)
Bovine insulin, by Merck Group (1 mentions)
Eif4e, by Cell Signaling Technology (1 mentions)
13 protocols using crystal violet powder
1
Immunocytochemistry and Comet Assay Protocol
2
Cell Wound Healing Assay with Taraxerol Acetate
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This assay was performed as previously described (17 (link)). Briefly, U87 cells (1×105 cells/ml) were seeded into a 6-well plate and incubated at 37°C for 24 h until a 95% confluent monolayer of cells was attained. Following 12 h of starvation, a 100-ml pipette tip was used to create a straight cell-free wound. Each well was washed three times with PBS to remove any cell debris, and then subjected to taraxerol acetate (0, 10, 50 and 150 µM) in DMEM. Subsequent to 48 h of incubation at 27°C, the cells were fixed and stained with 5% ethanol containing 0.3% crystal violet powder (Sigma-Aldrich) for 30 min, and images of randomly selected fields were captured under an IX71 inverted research microscope (Olympus Corporation). The number of cells that migrated into the scratched area were counted and the lengths of wound were determined by Image J software, version 1.46 (http://imagej.nih.gov/ij/ ).
3
Osteoblastic Cell Line MC3T3-E1 Characterization
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The mouse calvariae origin osteoblastic cell line (MC3T3-E1) subclone 14 (CRL-2594, highly differen-tiating) purchased from American Type Culture Collection (ATCC) Cell Bank (Manassas, VA, USA) was used as in vitro model. Cell culture reagents (alpha modified minimal essential medium (α-MEM), penicillin & streptomycin and fetal bovine serum (FBS)) were sourced from Gibco Laboratories (Grand Island, NY, USA). Ascorbic acid, β-glycerophosphate, and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium) dye were purchased from Sigma-Aldrich, USA. ALP activity assay kit was purchased from Abcam (ab83369) (USA). 5α-DHT and crystal violet powder were purchased from Sigma Aldrich, Germany. All other chemicals were sourced from the pharmaco-logy and cell culture laboratories of Universiti Kebangsaan Malaysia (UKM). All reagents and plastic wares used were trace element free and were analyzed for high purity grade.
4
Staining Protocol for Cell Visualization
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The chemicals used in this study included: glacial acetic acid, glycerol and saline obtained from El-Nasr Chemicals Co., crystal violet powder obtained from Sigma- Aldrich, methanol obtained from El Gomhouria Co. and chlorhexidine obtained from Arab Drug Company, Cairo, Egypt.
5
Quantifying Cell Proliferation via Crystal Violet
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Cell proliferation was determined using a Crystal Violet staining assay (Feoktistova et al., 2016 (link)). Briefly, 104 cells were cultured for 24, 48, 72 and 96 h under normal culture conditions following siRNA transfection. Following culture for the indicated time periods, cells were washed, fixed and stained in 1 ml of Crystal Violet staining solution [0.5% Crystal Violet powder (Sigma-Aldrich), 20% Methanol] for 20 min at room temperature with gentle agitation. Stained cells were solubilised in 1 ml 100% methanol/well and incubated with gentle agitation for 20 min at room temperature with lids on the plate. The optical density of each well was measured at 570 nm (OD570) using a plate reader. Average OD570 of control empty wells was subtracted from OD570 of wells containing cells.
6
Reagents and Antibodies for Cell Culture
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Cell culture reagents were purchased from Trace Biosciences (North Ryde, New South Wales, Australia) and Nunc (Roskilde, Denmark). Bovine insulin, methanol, calcium chloride, magnesium chloride, crystal violet powder and 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan were purchased from Sigma-Aldrich. Enhanced chemiluminescence (ECL) reagent was SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). The dual IGF1R/insulin receptor tyrosine kinase inhibitor (OSI-906; also referred to as linsitinib) was purchased from MedChem Express (Princeton, NJ) and the SphK inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKI-II) from Calbiochem [21 (link)]. Antibodies raised against phospho-Y1135/1136 IGFR1, IGFR1 beta chain, phospho-Ser473 AKT and total AKT, 4E-BP1 and eIF4E were purchased from Cell Signaling Technology (Beverley, MA). The antibody to detect SphK1 (ab16491) for western blots was purchased from Abcam and for SphK1 immunohistochemistry, from Abgent (AP7237c).
7
Cell Culture Protocol for Biological Assays
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Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640, Fetal Bovine Serum (FBS), and 0.25% trypsin were obtained from Gibco (California, USA). hygromycin B, MTT, crystal violet powder, and DMSO were purchased from Sigma (California, USA). The apoptosis detection kit was purchased from BD (State of New Jersey, USA). Bioluminescent substrates were purchased from Perkin Elmer (Waltham, USA); Matrigel was purchased from Corning (New York, USA). The BCA protein concentration assay kit was from Thermo (Waltham, USA). Anti-NLPR3 was from Novus (Colorado, USA, 1 : 500); anticyclin D1 and anticaspase-1 were purchased from Abcam (England, UK, 1 : 1000); anti-p–NF–κB, anti-NF-κB, anti-p-ERK 1/2, anti-ERK 1/2, anti-p-JNK, anti-JNK, anti-p-P38, anti-P38, PARP, Bcl-2, anti-GAPDH, and anti-β-actin were purchased from Cell Signal Technology (Boston, USA, 1 : 1000). The ECL chemiluminescence developer was purchased from Millipore (Massachusetts, USA). All other chemicals were of analytical grade.
8
Quantifying Macrophage Numbers via Crystal Violet
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Macrophage numbers were determined by crystal violet assay, modified from (27 (link)). Briefly, cell culture media was removed and cells were gently washed with water. Cells were incubated with 0.05% crystal violet solution (20 ml 95% ethanol, 80 ml water, and 0.5 g crystal violet powder (C6158, Sigma-Aldrich)) for 20 minutes at room temperature. Crystal violet solution was removed and cells were gently washed with water and allowed to air dry. After fully drying, 100% methanol was used to saturate the remaining crystal violet solution for 20 minutes. Absorbance was read at 595 nm using a DTX-880 Mutltimode plate reader (Beckman Coulter) or at 570 nm using a Synergy HTX Multi-Mode Reader and Gen5 software (BioTek) and plotted against a standard curve of known macrophage numbers.
9
Evaluating Dox and PD-0332991 Effects on GC Cells
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The GC cells were seeded in 12-well plates with 1 ml RPMI-1640 medium supplemented with 10% FBS at a density of 5×104 cells/well. Following incubation at 37.8°C for 24 h, the cells were treated with 20 nM Dox and/or 40 nM PD-0332991, and cultured for 24 h under a humidified atmosphere of 5% CO2 at 37.8°C. DMSO (0.1%) was used for the control group. The attached cells in 12 wells were stained with a crystal violet staining buffer [0.5% crystal violet powder (Sigma-Aldrich; Merck KGaA), 79.5% distilled H2O, 20% methanol], and incubated at room temperature for 1 h. The plates were subsequently washed with H2O three times and visualized using a digital camera (Canon, Inc.).
10
Clonogenic Survival Assay Protocol
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Established stable cell lines were plated in 6-cm culture dishes containing 4 ml of fresh medium with appropriate numbers in triplicate irradiated at 0–8 Gy on the day after cell plating and then incubated for 2 weeks. Cells were stained with crystal violet staining solution (0.5%) in 80 ml distilled water, 20 ml methanol and 0.5 g crystal violet powder (Merck, Whitehouse Station, NJ). Colonies containing more than 50 cells were counted and the surviving fraction was determined as the total number of colonies formed divided by the total number of cells plated multiplied by the plating efficiency, as determined in untreated cells.
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