Transwell assays were performed according to the manufacturer’s instructions. AGS cells in serum-free medium were seeded (5 × 104/mL/well) inside the chamber, while 500 μL culture medium containing 10% FBS was added into the outside of the chamber in each well of 24-well plate. After 24 h, non-invasive cells were removed from the upper surface of the membrane, while the invasive cells were fixed in methanol (10 min) and then stained with 0.1% crystal violet hydrate (Sigma, St. Louis, MO, United States; 30 min). After air-drying, the membrane was mounted on the slide and examined using microscope.
Crystal violet hydrate
Manufactured by Merck Group
Sourced in United States
Crystal Violet Hydrate is a laboratory chemical compound commonly used as a staining agent in various biological and analytical applications. It is a dark purple crystalline solid that dissolves in water and alcohol. Crystal Violet Hydrate is widely utilized in microbiology, histology, and cytology for staining and visualizing cellular structures.
Automatically generated - may contain errors
10 protocols using crystal violet hydrate
1
Quantifying Cell Invasion using Transwell Assay
2
Cell Invasion and Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell invasion and migration were detected with modified Boyden chamber (BD Biosciences, Sparks, MD, USA) assays. Briefly, three PC cell lines were seeded onto 8.0-µM pore size membrane inserts with 8.0 µM pores coated with matrigel (BD Biosciences) in 24-well plates with FBS-free growth media. 1640 plus 10% FBS was added to the bottom of the wells as a chemoattractant. 12-24 hrs later, cells that did not migrate were removed from the top side of the inserts with a cotton swab. Cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma) according to the manufacturer's instruction. The migratory cells were counted under a microscope at 20 x magnification. Cell images were obtained using a microscope (Nikon Microphot-FX, Japan), and counted in five random fields per insert. The migration assay was done in a similar fashion without matrigel. In more detail, we first used the same cell intensity (5×10^5/ml) to investigate different invasion ability of three normal PC cell lines. Because too high or too low cell population in invasion and migration assay is unfavorable for late result analysis in our pre-experiment, we seeded 1×10^5/ml of PANC-1 cells, 2.5×10^5/ml of BxPC-3 cells and 5×10^5/ml of Capan-2 cells in cell invasion and migration assayt. The results are presented as cells (actual number) migrated per field.
3
Transwell Chamber Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell chamber migration assays were performed using Nunc 24-well 8.0 μm pore transwell plates according to the manufacturer's instructions. Cells were plated at 5 × 104/ml in each well with 400 μl culture medium were added to the upper chamber. The lower chamber was added with 600 μl complete medium. After 36 h incubation, non-invading cells were removed from the upper surface of the membrane using a cotton-tipped swab. Then the invading cells were fixed in methanol for 10 min and stained with 0.1% Crystal Violet Hydrate (Sigma, St. Louis, MO, USA) for 5 min. The stained cells were counted as cells per field at 10× magnification. For TGF-β1 induction, cells were treated with TGF-β1 2 days before seeding and the upper chamber was added with TGF-β1.
4
Assessing Cell Invasion Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell invasion were assessed with modified Boyden chamber (BD Biosciences, Sparks, MD, USA) assays. Briefly, sg1-MSI2, sg2-MSI2 and scramble transfected PC cells (pretreated with EGF for 48 h) was seeded onto 8.0-μM pore size membrane inserts coated with matrigel (BD Biosciences) in 24 well plates with FBS-free growth media plus EGF. Growth media plus 10% FBS was added to the bottom wells as a chemoattractant. Twenty four hours later, cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma, St. Louis, MO, USA). The migratory cells were counted in five random fields per well. Each experiment was repeated three times.
5
Transwell Migration Assay of PC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, transfected PC cells (pretreated with TG or co-transfected with IRE1α) were plated in inserts that coated with matrigel (BD Biosciences, Sparks, MD, USA) in 24 well plates with FBS-free growth media. Growth media with 10% FBS was added to the bottom wells to generate a serum gradient. After 24 h, cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma). The migratory cells were counted in five random fields per well. The migration assay was done in a similar fashion without matrigel. Each experiment was repeated 3 times.
6
Cell Invasion Assay with Boyden Chamber
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell invasion was assessed with modified boyden chamber (BD Biosciences, Sparks, MD, USA) assays. Briefly, sg1-CRT, sg2-CRT and scramble-infected PC cells with or without EGF (50 ng/ml) treatment was seeded onto 8.0-μM pore size membrane inserts coated with matrigel (BD Biosciences) in 24-well plates with FBS-free growth media. Growth media plus 10% FBS was added to the bottom wells with or without EGF treatment as a chemoattractant. After 24 h, cells that did not migrate were removed from the top side of the inserts with a cotton swab. Cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma) according to the manufacturer’s instructions. The migration assay was carried out in a similar manner without matrigel. The migratory cells were counted under a microscope at × 20 magnification. Cell images were obtained using a microscope (Nikon Microphot-FX). Cells were counted in five random fields per insert. Results are expressed as cells migrated per field.
7
Evaluating Cell Migration in Pancreatic Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, FAM172AsiRNA and siRNAcontrol transfected AsPC-1 and BxPC-3 cells (pretreated with 20 µM of PD98059 for 2 h only once) was seeded onto 8.0-µM pore size membrane inserts (Corning Inc., NY, USA) coated with matrigel (BD Biosciences, Sparks, MD, USA) in 24-well plates with FBS-free growth media. Growth media plus 10% FBS was added to the bottom wells as a chemoattractant. 24 h later, cells that moved to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma-Aldrich, St Louis, MO, USA). The migratory cells were counted in five random fields per well. Results were expressed as cells migrated per field and repeated three times. The transwell assay was performed in a similar way without matrigel.
8
Cell Migration and Invasion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell migration assay, Capan‐2 and SW1990 cells were added to the top of chamber with growth media without FBS after transfection for 48‐72 hours or myoinositol (40 mg/mL) treatment for 48 hours. Growth media with 20% foetal bovine serum was added into the lower chamber as a chemoattractant. 24 hours later, the cells on the top of chamber were removed away Crystal Violet Hydrate (Sigma) under the instruction of manufacturer. And the cell invasion assay was conducted in an analogous method with the top of chamber pre‐coated with Matrigel (BD Biosciences). The images of cell migration and invasion were obtained via the microscope (Nikon Microphot‐FX) at 100× magnification; cells in every chamber were calculated under five random fields at 200× magnification. 5 × 106 cells/mL and 2.5 × 106 cells/mL of SW1990 cells and 2 × 106 cells/mL and 1 × 106 cells/mL of Capan‐2 cells were used to perform cell migration and invasion experiments, respectively. The experiments were repeated three times.
9
Quantifying Cell Migration Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brie y, transfected PC cells (pretreated with TG or co-transfected with IRE1α) were plated in inserts that coated with matrigel (BD Biosciences, Sparks, MD, USA) in 24 well plates with FBS-free growth media. Growth media with 10% FBS was added to the bottom wells to generate a serum gradient. After 24 h, cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma). The migratory cells were counted in ve random elds per well. The migration assay was done in a similar fashion without matrigel. Each experiment was repeated 3 times.
10
Quantifying Cell Migration Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brie y, transfected PC cells (pretreated with TG or co-transfected with IRE1α) were plated in inserts that coated with matrigel (BD Biosciences, Sparks, MD, USA) in 24 well plates with FBS-free growth media. Growth media with 10% FBS was added to the bottom wells to generate a serum gradient. After 24 h, cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma). The migratory cells were counted in ve random elds per well. The migration assay was done in a similar fashion without matrigel. Each experiment was repeated 3 times.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!