RPMI culture medium containing 0.3% agarose (Lonza, Basel, Switzerland) with LM05-shGFP or LM05-shNIK cells (4.0 × 104 cells/well) over a bottom layer of 0.6% agarose in RPMI culture medium were plated in each well of a 6-well plate and cultured for 3 weeks. Colonies were fixed with 4% paraformaldehyde-PBS (Fujifilm Wako Pure Chemical Corporation) for 1 h and stained with 0.005% crystal violet solution (Fujifilm Wako Pure Chemical Corporation) for 30 min. After removing the overdyed region with Milli-Q water, the colony images were acquired with a digital camera (Nikon Corporation, Tokyo, Japan), and colony numbers were calculated with ImageJ software (National Institutes of Health, MD, USA).
Crystal violet solution
Manufactured by Fujifilm
Sourced in Japan
Crystal violet solution is a laboratory reagent that is commonly used in various biological and chemical applications. It is a purple-colored dye that has the ability to stain cellular structures, particularly cell nuclei, making it useful for microscopic examination and analysis. The solution is prepared by dissolving crystal violet dye in a suitable solvent, typically water or an alcohol-based solution.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde pbs, by Fujifilm (1 mentions)
Digital camera, by Nikon (1 mentions)
Agarose, by Lonza (1 mentions)
Entellan new, by Merck Group (1 mentions)
Mscbm, by Lonza (1 mentions)
Cell counting kit 8, by Dojindo Laboratories (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Methanol, by Fujifilm (1 mentions)
Prism 8, by GraphPad (1 mentions)
11 protocols using crystal violet solution
1
Soft Agar Colony Formation Assay
2
Mouse Estrous Cycle Determination
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Estrous cycle was determined according to previous descriptions [21 (link)–23 (link)]. Briefly, the trunk of the mouse was gently pressed to immobilize it while the tail was lifted upward. The end of a sterile 200 μL-pipette tip filled with double distilled water (ddw, 50 μL) was placed at the opening of the vaginal canal, then the ddw was gently expelled into the canal and the mixture of water and internal fluid was withdrawn into the tip. Using the same tip, expulsion and aspiration procedures were repeated 4–5 times, then the fluid was dropped on glass slide. The wet smear was allowed to completely dry at room temperature for at least 1-h. The slides were incubated in 0.1 w/v% crystal violet solution (031–04852, FUJIFILM Wako Pure Chemicals, Japan) for 1 min, then washed twice in ddw, air-dried, and coverslipped with Entellan™ new (1.07961, Merck Millipore, Japan). Based on the cell morphology under a microscope with 40× magnification, the stage of the estrous cycle was identified as follows: proestrous, estrous, metestrous, or diestrous. Because the estrous cycle of a healthy mouse takes 4 days [22 (link)], the incidence of the estrous cycle was determined by vaginal smear cell morphology in 4-day blocks that met any of the following conditions: transition from proestrous to estrous, estrous, or transition from estrous to proestrous.
3
Colony Formation Assay for A549 and H1299 Cells
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The colony formation assay was performed as previously described (Yu et al., 2020 (link)). Briefly, A549 and H1299 cells were seeded in 6-well plates at a density of 500 cells/well and allowed to adhere for approximately 12 h. The cells were then treated with DMEM containing 25 μg/mL EF40 for 6 h, followed by culturing in normal medium for 10 days. Cells treated with normal medium were used as the controls. After treatment, cells were fixed with a methanol/acetone (v/v, 1:1) mixture for 10 min and stained overnight with 0.5% crystal violet solution (Wako). The number of colonies was counted, and the mean number of colonies was calculated from three independent experiments.
4
Clonogenic Survival Assay Protocol
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Cells were treated and irradiated in 96-well plates and were then re-seeded into 6-well plates at a density of 500 cells well−1. After attachment, cells were cultured in fresh medium for around 2 weeks. Forming colonies were then washed in cold PBS and were fixed with a pre-chilled methanol/acetone (v/v, 1:1) mixture for 10 min. Fixed cells were stained overnight with 0.1% crystal violet solution (Wako, Osaka, Japan). Numbers of colonies were counted and mean numbers of colonies were calculated from three independent experiments.
5
Quantifying Colony-Forming Capacity of hMSCs
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In a pilot study, we confirmed a positive correlation (P < 0.001, R = 0.992) between hMSC seeding density and capacity for CF. Because the CF measurement did not reflect the seeding density, the capacity for CF did not have an effect of the secretor factor and could measure quality of cells by CF measurement. The hMSCs at P4 were plated at 1.5 × 103 cells/T75 flask in MSCBM (Lonza Japan) for 14 days. The medium was changed at three- to four-day intervals. After embedding the plate in paraffin, the cells were stained by 1.0% crystal violet solution (Wako, Osaka, Japan) for 10 min. Aggregates of cells differentiated than 50 cells were counted as one colony, and we calculated the ratio of these colonies among all seeded cells.
6
Cell Viability and Colony Formation Assay
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Cell viability was assessed with a Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer's instructions. Cells (5 ×103 cells well−1) were seeded in a 96-well plate and allowed to adhere to the substratum overnight. They were then exposed to i-Ex loaded nanocomplexes as indicated. After washing with a fresh medium, the cells were incubated with CCK-8 solution for 2 h at 37°C. Absorbance at 450/690 nm was read on a microplate reader (Infinite M200 PRO; Tecan, Männedorf, Switzerland). A colony-forming assay was performed to evaluate long-term cytotoxicity of FRi-ExNC. Cells were seeded in a 12-well plate at a density of 200 cells well−1. After attachment, they were subjected to treatments for 6 h and allowed to foster in fresh medium for ≥10 days. The forming colonies were washed in cold PBS and fixed with a pre-chilled methanol/acetone (v/v, 1:1) mixture for 10 min. The fixed cells were stained overnight with a 0.1% crystal violet solution (Wako, Osaka, Japan). The colony-forming number was calculated as the mean number of colonies from three independent experiments.
7
Colony Formation Assay with DEPDC5
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Cells were plated at a density of 1 × 103 cells per well in 6-cm dishes and incubated at 37 °C with medium containing doxycycline or not. There were no significant differences in colony-forming capacity of HuH7 without pRetroX-TRE3G-DEPDC5 under normal medium with and without DOX. The cells were fixed by 100% methanol, and counterstained for nuclei with crystal violet solution (Wako) fourteen days later. The number of the stained cells was estimated by using ImageJ 1.51 software.
8
Cytotoxicity Evaluation of CAPE-based Compounds
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The long-term cytotoxicity effect of CAPE-PEG, CAPE-MotAb, DSPE-PEG-NHS, and MotAb on human cells was evaluated by colony-forming assay. Cells (500 per well) were seeded in 6-well plates and allowed to adhere to the substratum for overnight followed by 12-h treatment with CAPE-PEG, CAPE-MotAb, DSPE-PEG-NHS, and MotAb. The cells were cultured in normal medium with a regular change of medium every third day until colonies were formed. Colonies were washed thrice with cold PBS and fixed with methanol/acetone (1:1, v/v) at 4 °C for 10 min. Fixed colonies were again washed thrice with cold PBS, stained with 0.1% crystal violet solution (Wako, Osaka, Japan) overnight, de-stained with water, and left open for air drying. The plates were then subjected to photography, scanning by EPSON scanner, and colony counting.
9
Colony Formation Assay Protocol
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A total of 3 × 103 cells were seeded onto a D60 dish and cultured for 10 days. Cells were washed with PBS and were fixed with 4% PFA at RT for 10 min. Then, cells were washed with PBS and stained with 0.5% crystal violet solution (#031‐04852, Wako) for 30 min. After staining, cells were washed with tap water. The number of colonies was quantified by Image J.
10
Clonogenic Assay for Radiosensitization by SAS
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A clonogenic survival assay for determination of the radio-sensitizing effect of SAS was performed as previously described [14 (link)]. B16F10 cells were trypsinized, diluted, counted, and seeded into 60-mm dishes at densities of 100–10,000 cells/dish before being allowed to adhere in a 37°C incubator for 6 hours. Prior to irradiation, cells were incubated with SAS for 24 h. X-irradiation was performed with a linear accelerator (Primus MidEnergy, Siemens, Berlin, Germany). The dose rate was 2 Gy/min, determined using Fricke’s chemical dosimeter. The irradiated cells were then allowed to grow in a humidified 5% CO2 atmosphere at 37°C for 5 days before being fixed with methanol and stained with 0.05% crystal violet solution (Wako Pure Chemical Industries). Colonies that containing more than 50 cells were scored as surviving cells. The survival curves were then fitted to a linear–quadratic model using Origin Pro 7 data analysis software (Origin Lab Co., MA).
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