MatriGel (Corning, USA) was mixed with serum-free medium at 1:9 proportion and placed above Insert cell culture dish (Millipore, Germany) the night before and placed in a 37℃ incubator, then 1 × 105 cells in serum-free medium were placed above the dish with or without MatriGel for 24 and 48 h, the dishes were fixed with 4% paraformaldehyde and stained with Crystal Violet solution (Biosharp, China), the images were acquired using the Inverted microscope (Olympus, Japan).
Crystal violet solution
Manufactured by Biosharp
Sourced in China, United States
Crystal violet solution is a staining reagent commonly used in microbiology. It is a purple dye that binds to the cell walls of gram-positive bacteria, making them visible under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Biosharp (3 mentions)
Transwell chamber, by BD (2 mentions)
Matrigel, by BD (2 mentions)
Inverted microscope, by Zeiss (2 mentions)
Primovert, by Zeiss (2 mentions)
Matrigel, by Biosharp (1 mentions)
Matrigel, by Corning (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Beyoclick edu 594 cell proliferation assay kit, by Beyotime (1 mentions)
8 mm transwell, by Merck Group (1 mentions)
Transwell chamber, by Corning (1 mentions)
Matrix gel, by Corning (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Cck 8 solution, by Vazyme (1 mentions)
Axio observer d1 fluorescence microscope, by Zeiss (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
10 protocols using crystal violet solution
1
Cell Invasion Analysis on MatriGel
2
Colony Formation and Proliferation Assay
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For colony formation assay, cells were seeded in 6-well plates (4 × 103 cells / well) for two weeks. After fixation, colonies were dyed utilizing 0.1% crystal violet solution (21155722; Biosharp). 5-Ethynyl-2′-deoxyuridine (EdU) staining was conducted with BeyoClick™ EdU-594 cell proliferation assay kit (C0078S; Beyotime, China) following the manufacturer’s instructions. The percentage of EdU-positive cells was calculated.
3
Cell Migration Assay by Transwell
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A transwell assay was also used to assess cell migration. Initially, 2 × 104 cells seeded in the upper chambers of every 8 mm transwell (Millipore, Billerica, MA, United States) were irradiated with 4 Gy of X-rays. The cells in the upper chamber were incubated in serum-free medium, whereas medium with 10% serum was added to the lower chamber. After removing cells on the upper surface of the filter after 48 h, cells attached to the lower chamber were fixed with 4% paraformaldehyde (Biosharp) for 30 min, stained with 0.1% crystal violet solution (Biosharp)for 30 min. Three areas were selected randomly for photography and calculation using an inverted microscope (Carl Zeiss).
4
Transwell Assay for Cell Migration and Invasion
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Cell migration was examined using transwell chambers (0.8 μm 24-well plates, Corning, USA). Cells (1 × 104) were suspended in serum-free medium (200 μl) and seeded into the chambers before 500 μl of medium containing 10% FBS was added to the bottom. After a 24 h incubation, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet solution (Biosharp, USA). Then the cells were observed by microscopy by randomly choosing six fields to count the number of migrated cells. For cell invasion analysis, matrix gel (Corning) was added to the transwell chambers before cell seeding; all other procedures were the same.
5
Cell Proliferation Assessment Protocols
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Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and colony‐forming assays. A total of 5 × 103 transfected cells in 100 μL medium per well were added to a 96‐well plate for 4, 8, 24, 48, and 72 hours. At the indicated times, 10 μL (at a concentration of 10%) CCK-8 solution (Vazyme, Nanjing, China) was added to each well and incubated for 1 hour at room temperature. The absorbance was assessed at a 450 nm wavelength under a plate reader (BioTek ELx800). For the Colony‐forming assays, transfected A549 and H1299 cell lines were seeded (1 × 103 cells/well) into six-well plates with 2 mL complete medium and divided into an si-NC and si-UBE2T groups. The colonies were fixed with 4% methanol (Solarbio) after 7–10 days of culture and then stained with 0.1% crystal violet solution (Biosharp, China). After 15 min, the cells were washed gently with PBS 3 times and then air‐dried. Finally, the colony‐forming units (consisting of ≥50 cells) were observed under an inverted microscope (ZEISS Primo Vert) and counted using ImageJ software. All experiments were performed in triplicate.
6
Clonogenic Assay for Radiation Sensitivity
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Cells (1 × 103) were seeded into 60 mm cell culture dishes (NEST, Wuxi, China), treated with X-rays at doses of 0, 2, 4, or 6 Gy, and then cultured for 14 days to allow colonies to grow. Then, colonies were fixed with 4% paraformaldehyde (Biosharp), stained with 0.1% crystal violet solution (Biosharp), and counted using ImageJ software (National Institutes of Health, Bethesda, MD, United States).
7
Transwell Assay for Cell Migration and Invasion
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Medium (600 μL) containing 20% FBS was added to the lower chamber of the Transwell (Corning Inc., Corning, NY, USA), and 200 μL of serum-free medium was added to the upper chamber. SKOV3 cells (3 × 104) or A2780 cells (5 × 104) were seeded into Transwell chambers without or with Matrigel (BD Biosciences, San Jose, CA, USA) (60 μL/well; medium/Matrigel ratio, 7∶1) to conduct migration or invasion assays, respectively. Following 24 to 72 h of cultivation, the chamber was fixed with 4% paraformaldehyde (BioSharp, Hefei, Anhui, China) and then stained with 0.1% crystal violet solution (BioSharp). Cells were wiped away from the upper surface of the chamber using a cotton swab, and images of the lower surface of the chamber were acquired for randomly selected areas using an Axio Observer D1 fluorescence microscope (Carl Zeiss).
8
Transwell Cell Migration Assay
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The transwell chambers (BD, USA) were covered by Matrigel (BD, USA) for 3 h in incubator and arranged in the 24-well plate with medium containing 10% FBS. The HeLa cells were preincubated with or without RES and PUR, then, seeded into each chamber at concentration of 2 × 104/200 μL serum-free medium. The cells were incubated in the incubator for 24 hours and were fixed by 5% paraformaldehyde and stained with 0.1% crystal violet solution (Biosharp, China). The cells were scrubbed with cotton swab in the upper of chamber and randomly selected to photograph in the bottom of chamber. Finally, the number of cells was analyzed with ImageJ software.
9
Transwell-based Invasion Assay for EC Cells
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The in vitro invasion ability of the EC cell line was analyzed as previously described (35 (link)). In brief, we used 8-µm pore Transwell cell culture chambers (Corning, Inc.) coated with 30 µl specific Matrigel (BD Biosciences) which served as a reconstituted basement membrane for the invasion assay. Cells (2×104) were plated in upper chamber. The cells were then resuspended in 200 µl DMEM medium without FBS and placed in the top compartment of the Transwell chambers. Then, optimal volume of standard culture medium (10% FBS), which was regarded as chemoattractant, was added into the lower chamber and the staining was performed at room temperature. After 24 h, EC cells on the upper surface were carefully removed by a clean cotton swab. After removing the non-invasive cells, the cells at the bottom of the lower chamber were harvested and fixed in 4% PFA (PBS; Solarbio) for 30 min. After PBS washing, the cells were stained with 0.1% crystal violet solution (Biosharp) for ~30 min. After rinsed in PBS for 10 min, the total number of invasive cells was accurately detected under an Olympus fluorescence microscope (Olympus Corp.). The total number was counted by using AIS software.
10
Colony Formation Assay on MCF-7 Cells
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MCF-7 cells were plated into 6-well plates (200 cells/well) and cultured for 14 day to form colony. After 14 day, 0.1% crystal violet solution (Biosharp, Beijing, China) was used to fix and stain colonies for 30 min. Next, the number of colonies was calculated.
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