For stably transfected cell lines, 100 cells were cultured in 10‐cm dishes for 14 days. For transiently transfected cells, 1 × 105 cells were cultured in a 10‐cm dish and selected with G418 (1.2 mg/mL) for 14 days. The resulting colonies were fixed with 10% buffered formalin, stained with 0.05% toluidine blue solution (pH 7.0) (Wako, Osaka, Japan) and quantified.
Toluidine blue solution
Manufactured by Fujifilm
Sourced in Japan
Toluidine Blue Solution is a laboratory reagent used in various staining and analytical procedures. It is a metachromatic dye that can bind to acidic components in cells and tissues, allowing for the detection and visualization of specific structures or molecules. The solution's core function is to provide a staining agent for microscopic examination and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Biocoat growth factor reduced matrigel invasion chamber, by BD (2 mentions)
Buffered formalin, by Fujifilm (1 mentions)
Xylene, by Fujifilm (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Oct compound, by Fujifilm (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Glass slide, by Matsunami (1 mentions)
4 protocols using toluidine blue solution
1
Cell Colony Formation Assay
2
Matrigel Invasion Assay for Cell Migration
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An invasion assay was performed using a BD BioCoat Growth Factor Reduced Matrigel Invasion Chamber (354483, BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Cells were starved in 1640 medium without FBS for 24 h and then plated on the upper insert at 2.5×104/well and incubated in 1640 medium with FBS in 20% or 0.1% oxygen cell chambers, respectively. After a 24-h incubation, the non-invading cells remained on the upper surface of the membrane in each insert were gently removed. Cells that had penetrated through to the bottom surface of the membranes were fixed in 100% methanol and stained with 0.05% Toluidine Blue Solution (206-14555, Wako Pure Chemical Industries Ltd.). For each experiment, the number of cells in seven randomly chosen fields of each filter was quantified, and these experiments were independently performed at minimum of three times.
3
Cell Invasion Assay with Matrigel
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The conventional invasion assay was performed using a BD BioCoat Growth Factor Reduced Matrigel Invasion Chamber (354483, BD Biosciences, NJ, USA) as described previously42 (link). In brief, cells, which were plated on the upper insert at 1 × 105/well with or without nanoparticles, were incubated for 24 h. The invaded cells on the under surface of the membrane were fixed after removing the cells on the upper surface of the membrane in each insert. The number of invaded cells, which were stained with 0.05% Toluidine Blue Solution (206-14555, Wako), was manually counted by microscopy.
4
Quantification of Mast Cells in Ear Tissue
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The left ears were embedded in OCT compound (Sakura Finetek, Tokyo, Japan), frozen and then sectioned to 10 μm with the Leica CM3050S cryostat (Leica, Wetzlar, Germany). Sections were mounted on glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan), dried overnight, washed in water to remove the OCT compound, and then stained with 0.05% Toluidine Blue Solution (pH 4.1; Wako Pure Chemical Industries, Osaka, Japan) for 10 min. After staining, sections were washed briefly in water, subjected to differentiation and dehydration 3 times with 95% ethanol, and another 3 times with 99% ethanol, cleared in xylene (Wako) 3 times, and then sealed in Entellan® new cover slipper for microscopy (Merck KGaA, Darmstadt, Germany). After air-drying, images of the sections were acquired in the same orientation under a microscope and mast cells were counted under blinded conditions. Six fields of view were randomly selected per sample, and the average of these fields was taken as the number of cells.
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