Metformin (D150959), CQ (C6628), MDC (30432), 3-MA (M9281), 4',6-diamidino-2- phenylindole (DAPI; D9542), antibodies against anti-β and LC3, anti-rabbit secondary antibody, anti-mouse secondary antibody and Cy3-labeled rabbit secondary antibody were purchased from Sigma (St. Louis, MO, USA). Antibodies against p-Stat3 (Y705), Stat3, cyclin D1, LC3, PCNA and PARP were purchased from Cell Signaling (Beverly, MA, USA); antibodies against p-AMPK, p-mTOR, Beclin-1, Bcl-2, p62 and Bax were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-phospho-Histone H3 (Ser10) antibody was purchased from Upstate (Millipore, Billerica, MA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Gibco (Life Technologies Gibco/BRL, New York, NY, USA). Crystal violet staining and JC-1 were obtained from Beyotime Institute of Biotechnology (Shanghai, China). ApopTag plus peroxidase in situ apoptosis detection kit was obtained from Millipore. AO was obtained from Poly-Sciences (Warrington, PA, USA). Stat3, AMPK, Beclin-1 and Atg5 siRNAs were purchased from Shanghai GenePharma (Shanghai, China). Lipofectamine 2000 was bought from Invitrogen (Carlsbad, CA, USA).
Crystal violet staining
Manufactured by Beyotime
Sourced in China
Crystal violet staining is a laboratory technique used for the visualization and identification of bacterial cells. It is a primary stain that binds to the peptidoglycan layer of the bacterial cell wall, turning the cells purple in color. This staining method is a core component of the Gram staining procedure, which is widely used for the differentiation of Gram-positive and Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti rabbit secondary antibody, by Merck Group (1 mentions)
Atg5 sirna, by GenePharma (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Apoptag plus peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Cy3 labeled rabbit secondary antibody, by Merck Group (1 mentions)
Stat3, by GenePharma (1 mentions)
Beclin1, by GenePharma (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Beclin 1, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Transwell insert, by Corning (1 mentions)
Matrigel, by BD (1 mentions)
Transwell chamber, by Corning (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Matrigel invasion chamber, by Corning (1 mentions)
Cck 8 assay kit, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
14 protocols using crystal violet staining
1
Autophagy Modulation in Cancer Therapeutics
2
Transwell Assay for Cell Migration and Invasion
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Cell migration and invasion assays were performed in transwell tests. For cell migration test, 5 × 105 cells in 200 μl of FBS-free RPMI 1640 was added to upper chamber of transwell inserts with diameter of 6.5 mm and pore size of 8 μm (Corning, USA). In the 24-well plate with transwell inserts, 600 μl of RPMI 1640 culture medium containing 10% FBS and 1% PS was added to the lower part of the chamber. For invasion tests, a Matrigel layer was prepared on the bottom of the upper chambers before cell seeding. After incubation at 37 °C, 5% CO2 for 24 h, the upper chamber was cleaned gently with cotton swabs, fixed with 4% paraformaldehyde, stained with crystal violet staining (Beyotime, China), and washed 3 times with water. The cells migrated to the other side of the filter was imaged using an inverted microscope (Nikon, Shanghai). The quantification of cells were performed by cell counting from 8 images of the same transwell test. Duplicate of transwell test were done for each condition. 10 μg/ml exosomes were used for functional assays.
3
Colony Formation Assay Protocol
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Cells (2 × 104) were seeded into 6-cm plates and incubated for a 14-day period. Afterwards, methanol was added to fix colonies for a 20-min period (Beyotime, Nanjing, China), followed by 0.1% crystal violet staining (Beyotime), assessment, and count of colonies under the microscope (Olympus, Tokyo, Japan).
4
Transwell Assay for Cell Invasion and Migration
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Cell invasion assays were performed using transwell chambers (Corning, NY, USA) precoated with diluted Matrigel (1 : 8 dilution; BD Biocoat, Corning), while cell migration assays were completed without Matrigel. A total of 40,000 cells in 200 μL of serum-free DMEM were seeded in the upper chambers, and the lower chambers were filled with DMEM containing 20% FBS. After 48 h of incubation at 37°C, the cells migrated or invaded into the lower surface were fixed with 4% paraformaldehyde (Beyotime Biotechnology) and visualized using crystal violet staining (Beyotime Biotechnology). The images of cells were captured at 40x using a light microscope, and five random fields were counted in each chamber. Three independent experiments were performed.
5
Evaluating CRC Cell Migration and Invasion
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A wound‐healing assay assisted in assessing CRC cell migration ability. Cells were seeded in a 6‐well plate and cultured overnight. A 200 μL pipette tin was employed to scratch a straight line, which was then cultured in a fresh medium. Migrated cells were detected with a microscope (Leica) at 0, 24, 48, 72, 96 and 120 h. Image‐pro plus 6.0 software was used for the analysis of the images. Next, a transwell assay served for the evaluation of the cell invasion. Briefly, cells seeded in a matrigel invasion chamber (Corning) received 48 h of culturing at 37°C. Invaded cells received crystal violet staining (Beyotime). A microscope illustrated related images.
6
Cell Viability Assay of SHP099 on SMSCs
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The cell viability of SHP099 on SMSCs was assessed using a CCK-8 assay kit (Thermo Fisher Scientific) according to the instructions. Cells were cultured in 96-well plates and incubated with different concentrations of SHP099 for 1 week. CCK-8 reagents were added to each well for 3 h at 37 °C. At least 5 measurements in each group were performed by detecting the absorbance at 450 nm. And crystal violet staining (Beyotime Biotechnology) was performed according to the instructions.
7
Colony Formation Assay of 4MOD-Treated Cells
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1 × 103 cells/well were planted overnight in 6-well plates (Corning, NY, USA). After treatment with or without 4MOD for 48 h, cells were incubated in a 5% CO2 humidified incubator for 2–3 weeks. When the colonies appeared, cells were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) and stained with crystal violet staining (Beyotime, Shanghai, China). An inverted microscope was used to count colonies.
8
Cell Migration Assay Protocol
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Cell suspensions were prepared using serum-free medium and diluted to 105 cells/ml. Subsequently, 200 µl of the cell suspension was added to the supporting chambers of 24-well plates (Corning, Inc.). The medium in the lower well was Dulbecco's modified Eagle's medium/F12 (HyClone; GE Healthcare Life Sciences) containing 10% foetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Following incubation for 24 h, the cells were fixed with paraformaldehyde (Bioshap), subjected to crystal violet staining (Beyotime Institute of Biotechnology) at room temperature for 30 min and imaged using an inverted microscope (Eclipse Ci; Nikon Corp.). ImageJ software (version 1.6; National Institutes of Health) was used to analyse the number of cells in each image and perform statistical analysis.
9
Evaluating Cell Invasion Capacity
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The invasion capacity of the cells was evaluated using a Matrigel invasion chamber. First, cell inserts (Costar; Corning Co., Corning, USA) were coated with 100 μL of basement membrane matrix (1:8 dilution; BD Biosciences, Franklin Lakes, USA) and placed in a 24well plate at 4°C overnight. The chamber for the migration test was not pre-coated with Matrigel. Invasion experiments were performed using 6×10 5 HTR8/SVneo cells. A total of 150 μL of cell suspension was placed in the upper chamber, and 750 μL of medium containing 10% fetal bovine serum was placed in the lower chamber. After 24 h of culture, the cells on the apical side were removed with cotton swabs, while cells on the basal side were rinsed with PBS, fixed in methanol, and subjected to crystal violet staining (Beyotime). Finally, the invaded or migrated cells were observed using an optical microscope (Life Technologies) and the number of cells was counted. The experiment was repeated three times for each group.
10
Transwell Invasion Assay Protocol
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Cell invasion behavior was evaluated by using a transwell assay. The 24-well
transwell chambers (8μm pore size; Millipore) coated with matrigel were
prepared, and the transfected OC cells at 4 ×104 cells/ml density was
added onto the top chamber with 200 µl serum-free RPMI-1640 medium. Meanwhile,
500 µl RPMI-1640 medium containing 10% FBS was added to the basolateral chamber.
After being cultured for 48 h, the cells invaded into the basolateral chamber
were treated with 4% paraformaldehyde solution (Sigma) for 20 min at 37°C,
followed by crystal violet staining (Beyotime Biotechnology) for 15 min at 37°C.
In contrast, the cells in the apical chamber were wiped with cotton swabs. The
staining cells were counted for five visual fields (magnification, ×200) with an
optical microscope (Olympus, Tokyo, Japan). Each experiment was performed in
triplicate.
transwell chambers (8μm pore size; Millipore) coated with matrigel were
prepared, and the transfected OC cells at 4 ×104 cells/ml density was
added onto the top chamber with 200 µl serum-free RPMI-1640 medium. Meanwhile,
500 µl RPMI-1640 medium containing 10% FBS was added to the basolateral chamber.
After being cultured for 48 h, the cells invaded into the basolateral chamber
were treated with 4% paraformaldehyde solution (Sigma) for 20 min at 37°C,
followed by crystal violet staining (Beyotime Biotechnology) for 15 min at 37°C.
In contrast, the cells in the apical chamber were wiped with cotton swabs. The
staining cells were counted for five visual fields (magnification, ×200) with an
optical microscope (Olympus, Tokyo, Japan). Each experiment was performed in
triplicate.
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