C2C12 cells were treated with the indicated doses of dapagliflozin for 24 h in the hyperglycemic condition (final concentration of glucose: 25 mM). Following the treatment, cells were reseeded in a 96-well plate and exposed to hypoxia at indicated times. Cell proliferation assay was performed by using an MTS reagent kit (Promega, Madison, WI) for 2 h. Viable cell numbers were measured with a spectrophotometric microplate reader (BioTek Instruments, Winooski, VT) at a wavelength of 490 nm. For crystal violet staining, C2C12 cells were seeded in a 24-well plate and treated with indicated doses of dapagliflozin for 24 h. Following 24 h exposure to hypoxia, cells were fixed by 5 min incubation with 4% paraformaldehyde (PFA) and stained for 30 min with 0.05% crystal violet (Beyotime, Shanghai, China).
Crystal violet
Manufactured by Beyotime
Sourced in China, United States, Japan, Germany
Crystal violet is a synthetic dye commonly used in laboratory settings. It is a dark purple crystalline solid that is soluble in water and alcohol. Crystal violet is often used as a staining agent in various biological and medical applications, including microscopy and cell identification.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (229 mentions)
Paraformaldehyde (pfa), by Beyotime (144 mentions)
Matrigel, by Corning (102 mentions)
Transwell chamber, by Corning (94 mentions)
Paraformaldehyde (pfa), by Merck Group (47 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (28 mentions)
Transwell plate, by Corning (27 mentions)
Inverted microscope, by Olympus (25 mentions)
C0121, by Beyotime (24 mentions)
Methanol, by Beyotime (23 mentions)
Transwell chamber, by BD (23 mentions)
Transwell chamber, by Merck Group (23 mentions)
Paraformaldehyde (pfa), by Sangon (19 mentions)
Upper chamber, by Corning (19 mentions)
Matrigel matrix, by BD (19 mentions)
Optical microscope, by Olympus (15 mentions)
Transwell insert, by Corning (15 mentions)
24 well transwell chamber, by Corning (15 mentions)
Paraformaldehyde (pfa), by Solarbio (14 mentions)
Microplate reader, by Thermo Fisher Scientific (14 mentions)
1 732 protocols using crystal violet
1
Dapagliflozin Effects on C2C12 Cell Proliferation
2
Cell Proliferation and Migration Assays
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Cellular growth curves and colony-forming assays were used to evaluate cell proliferation rates. Briefly, cells were seeded, cultured, and incubated with the CCK-8 kit (Dojindo, Japan) reagent, and the optical density (OD) value was analyzed using a Synergy HT multi-mode reader (BioTek, USA) at 450 nm. A plate colony-forming assay was performed by seeding 200 cells/well in a culture dish. The cells were fixed, stained with crystal violet (Beyotime, China), and counted under a microscope (> 50 clones validated; Leica) after 14 days of culture.
Cell migration was evaluated by wound healing and transwell assays. Twenty-four hours after cell seeding and culturing, a scratch was made using a 200 μL sterile pipette tip. The cells were cultured in FBS-free medium, and wound confluence was observed after 24 and 48 h. The transwell assay was also used to confirm cell migration. A 2 × 104 single-cell suspension was added into the upper chambers of 24-well transwell plates (Corning Life Science), and 0.1% FBS-containing medium was added to the lower chambers. The migrated cells were fixed, stained with crystal violet (Beyotime), and counted under a microscope after 24 h incubation.
Cell migration was evaluated by wound healing and transwell assays. Twenty-four hours after cell seeding and culturing, a scratch was made using a 200 μL sterile pipette tip. The cells were cultured in FBS-free medium, and wound confluence was observed after 24 and 48 h. The transwell assay was also used to confirm cell migration. A 2 × 104 single-cell suspension was added into the upper chambers of 24-well transwell plates (Corning Life Science), and 0.1% FBS-containing medium was added to the lower chambers. The migrated cells were fixed, stained with crystal violet (Beyotime), and counted under a microscope after 24 h incubation.
3
Evaluating Cell Viability, Colony Formation, and Migration
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Cell viability was assessed utilizing a Cell Counting Kit-8 assay (CCK-8, APExBIO) according to the manufacturer’s instructions. The cell growth curve was plotted as a function of time and absorbance value (OD) at 450 nm.
For the colony formation assays, approximately 1000 cells were seeded onto 6-well plates for 10 days. Next, the colonies that were formed were washed with PBS, fixed with 4% paraformaldehyde (Beyotime) and stained using 0.1% crystal violet (Beyotime). Next, the images were captured using a digital camera (Nikon Corporation; Tokyo, Japan).
For transwell migration assays, GC cells within the serum-free medium were seeded into the upper chambers (Corning Incorporated; USA). The lower chamber contained medium with 10% FBS as a chemoattractant. After incubating for 12–48 h, the cells that had not migrated through the pores in the upper chambers were removed manually using a cotton swab. Cells that had migrated through the membrane were fixed in 4% paraformaldehyde (Beyotime), and stained using 0.1% crystal violet (Beyotime).
For the colony formation assays, approximately 1000 cells were seeded onto 6-well plates for 10 days. Next, the colonies that were formed were washed with PBS, fixed with 4% paraformaldehyde (Beyotime) and stained using 0.1% crystal violet (Beyotime). Next, the images were captured using a digital camera (Nikon Corporation; Tokyo, Japan).
For transwell migration assays, GC cells within the serum-free medium were seeded into the upper chambers (Corning Incorporated; USA). The lower chamber contained medium with 10% FBS as a chemoattractant. After incubating for 12–48 h, the cells that had not migrated through the pores in the upper chambers were removed manually using a cotton swab. Cells that had migrated through the membrane were fixed in 4% paraformaldehyde (Beyotime), and stained using 0.1% crystal violet (Beyotime).
4
Colorimetric Assays for Cell Proliferation and Migration
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For the colony formation assays, about 1,000 cells were cultivated in 6-well plates for 7-10 days. Colonies formed were washed with PBS, fixed with 4% paraformaldehyde (Beyotime, Beijing, China) and stained with 0.1% crystal violet (Beyotime, Beijing, China). Colonies containing more than 50 cells for each well were counted.
For transwell migration assays, cells were seeded into the upper chambers (Corning Incorporated, USA) in serum-free RPMI 1640 and the lower chamber was filled with RPMI 1640 containing 10% FBS. After incubation for 12-48 h, cells which had migrated through the membrane were fixed with 4% paraformaldehyde (Beyotime, Beijing, China) and stained with 0.1% crystal violet.
For transwell migration assays, cells were seeded into the upper chambers (Corning Incorporated, USA) in serum-free RPMI 1640 and the lower chamber was filled with RPMI 1640 containing 10% FBS. After incubation for 12-48 h, cells which had migrated through the membrane were fixed with 4% paraformaldehyde (Beyotime, Beijing, China) and stained with 0.1% crystal violet.
5
Colony Formation and Cell Migration Assays
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For colony formation assay, the GBC-SD or SGC-996 LKB1 and their control cells were seeded in 12-well plates (3x10 3 cells/well). The cells were incubated at 37˚C for 7 days and then fixed with with 10% neutral formalin for >4 h. The cells were dyed with crystal violet (Beyotime, Haimen, China). The cells were photographed under a microscope (Olympus, Tokyo, Japan).
Cell migration assay. For cell migration experiments, 2x10 5 cells were seeded into the upper chamber of a polycarbonate Transwell in serum-free DMEM. The lower chamber was supplemented with DMEM containing 20% FBS as chemoattractant. The cells were incubating for 24 h and the chamber was fixed with 10% neutral formalin for >4 h. The cells were dyed with crystal violet (Beyotime). The cells were then counted under a microscope (Olympus) and the cell number is expressed as the average number of the cells in each field.
Cell migration assay. For cell migration experiments, 2x10 5 cells were seeded into the upper chamber of a polycarbonate Transwell in serum-free DMEM. The lower chamber was supplemented with DMEM containing 20% FBS as chemoattractant. The cells were incubating for 24 h and the chamber was fixed with 10% neutral formalin for >4 h. The cells were dyed with crystal violet (Beyotime). The cells were then counted under a microscope (Olympus) and the cell number is expressed as the average number of the cells in each field.
6
Anchorage-Independent Growth Assay
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U251 cells were cultured in 12 wells and treated with 0, 50, and 100 nM TPL, and one well transfected with pcDNA3.1-PROX1 plasmid and 100 nM TPL and harvested as single-cell suspension. We resuspended 1 × 104 cells in 500 μl of a 1:1 mix of medium and Matri-gel (Biozellen), and 100 μl drops were placed on precooled 24-well plates and allowed to solidify for 5 minutes at 4°C. Another 1ml of fixing solution was added for 15 minutes, and then 1ml of the medium was added and cultured in an incubator for 1-2 weeks. Plates were stained with crystal violet (Beyotime) and washed to remove excess crystal violet. The number of colonies was scored with Image J.
7
Crystal Violet Staining of HUVEC
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THP-1 cells differentiation and transfection were performed as mentioned above. HUVEC was seeded in chambers (#14112, PET Polyester, 0.4 μm, 24 mm, Labselect, Anhui, China) and transduced the next day for 24 h. The procedure is detailed in Supplemental 1 . HUVEC was fixed for 15 min with 4% paraformaldehyde at 23 °C. Subsequently, chambers were washed by submersion in a water bath, followed by permeation using anhydrous methanol for 20 min at 23 °C. For crystal violet staining, chambers were aspirated and 0.5% crystal violet (Beyotime, Shanghai, China) was added for the cells from the bottom of the chamber. After 10 min at 23 °C, crystal violet was removed, and the chambers were thoroughly washed thrice using PBS. Images were acquired under a microscope (Olympus, Tokyo, Japan) and analyzed using the Image J software (NIH, Bethesda, MD, USA).
8
Melanoma Cell Proliferation and Migration Assays
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For the Proliferation assay, 500 melanoma cells were seeded in 6-well plates and incubated at 37 °C. After two weeks, cells were fixed and stained with crystal violet (Beyotime, Shanghai, China) for 30 min.
The migration assays were conducted using Transwell™ filter, a modified two-chamber plate with a pore size of 8 μm. The treated melanoma cells were seeded in serum-free medium in the upper chamber and the medium with 10% FBS was added to the lower compartment. After 12 h of incubation at 37 °C, the melanoma cells in the upper chamber were carefully removed using a cotton swab, and the cells in the lower compartment were stained with crystal violet (Beyotime, Shanghai, China) for 30 min.
The migration assays were conducted using Transwell™ filter, a modified two-chamber plate with a pore size of 8 μm. The treated melanoma cells were seeded in serum-free medium in the upper chamber and the medium with 10% FBS was added to the lower compartment. After 12 h of incubation at 37 °C, the melanoma cells in the upper chamber were carefully removed using a cotton swab, and the cells in the lower compartment were stained with crystal violet (Beyotime, Shanghai, China) for 30 min.
9
Transwell Assay for Cell Migration and Invasion
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For cell migration experiments, 2×105 HCCLM3 miR-369 mimic or Huh7 cells miR-369 mimic and their control cells were seeded into the upper chamber of a polycarbonate transwell in serum-free DMEM medium. The lower chamber was added with DMEM medium containing 20% FBS as chemoattractant. The cells were incubating for 16 hours and the chamber was fixed with 10% neutral formalin for more than 4 hours. The cells were dyed with crystal violet (Beyotime). The cells were then counted under a microscope (Olympus) and the cell number is expressed as the average number of the cells in each field.
For cell invasion experiments, 2×105 HCCLM3 miR-369 mimic/sponge or Huh7 cells miR-369 mimic/sponge and their control cells were seeded into the upper chamber of a polycarbonate transwell in serum-free DMEM medium. The lower chamber was added with DMEM medium containing 20% FBS as chemoattractant. The cells were incubating for 36 hours and the chamber was fixed with 10% neutral formalin for more than 4 hours. The cells were dyed with crystal violet (Beyotime). The cells were then counted under a microscope (Olympus) and the cell number is expressed as the average number of the cells in each field.
For cell invasion experiments, 2×105 HCCLM3 miR-369 mimic/sponge or Huh7 cells miR-369 mimic/sponge and their control cells were seeded into the upper chamber of a polycarbonate transwell in serum-free DMEM medium. The lower chamber was added with DMEM medium containing 20% FBS as chemoattractant. The cells were incubating for 36 hours and the chamber was fixed with 10% neutral formalin for more than 4 hours. The cells were dyed with crystal violet (Beyotime). The cells were then counted under a microscope (Olympus) and the cell number is expressed as the average number of the cells in each field.
10
Single-Cell Colony Formation Assay
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Colony formation assay was used to assess single cell proliferation ability.
Firstly, cells were cultured at a density of 1 × 104 cells per well
in 6-well plates for 1 week. Next, cells were fixed by 4% paraformaldehyde
(Sigma) for 15 min at room temperature. Then, cells were stained by 0.5% crystal
violet (Beyotime) for 15 min at room temperature. Finally, after washing with
water, colonies were observed under the optical microscope (Leica), and the
number of colonies was counted.
Firstly, cells were cultured at a density of 1 × 104 cells per well
in 6-well plates for 1 week. Next, cells were fixed by 4% paraformaldehyde
(Sigma) for 15 min at room temperature. Then, cells were stained by 0.5% crystal
violet (Beyotime) for 15 min at room temperature. Finally, after washing with
water, colonies were observed under the optical microscope (Leica), and the
number of colonies was counted.
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