To obtain BAL fluid, the tracheas of anesthetized mice were exposed and cut just below the larynx. A polyurethane flexible intravenous catheter tube [0.7-mm outer diameter, 19 mm in length, and attached to a blunt 24-guage needle (Becton Dickinson and Company, Franklin Lakes, New Jersey, USA)] was placed into the trachea, after which the lung lavage was done for 1 time with 800 μl of sterile cold PBS. The samples were centrifuged at 5,000 rpm for 5 min at 4°C. The supernatants were decanted and immediately frozen at −70°C. Cell pellets were washed twice in PBS. The total cell number was counted using a hematocytometer. The BAL fluid cells were attached for 5 min at 650 rpm with cytospin-X (Hanil, Gimpo, Korea). Adhered cells were stained with Diff-Quick solution (Sysmex Co., Kobe, Japan) and cells were differentially counted according to conventional morphological criteria. At least 100 cells per slide were counted to obtain differential leukocyte counts.
Diff quick solution
Manufactured by Sysmex
Sourced in Japan
Diff-Quick solution is a rapid staining system used for the differential staining of blood smears. It is a three-step staining procedure that differentiates white blood cells and provides a clear visualization of cellular morphology. The solution contains a combination of dyes that stain the nucleus, cytoplasm, and other cellular components, enabling efficient and consistent staining of blood samples.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer a1, by Zeiss (3 mentions)
Biocoat matrigel invasion chamber kit, by BD (2 mentions)
Matrigel, by Corning (1 mentions)
Axio imager 2, by Zeiss (1 mentions)
Rbc lysis buffer, by Merck Group (1 mentions)
Nucleocounter, by ChemoMetec (1 mentions)
Micro 12tm, by Hanil (1 mentions)
6 well plate, by Corning (1 mentions)
Optical microscopy, by Olympus (1 mentions)
15 protocols using diff quick solution
1
Bronchoalveolar Lavage Fluid Extraction
2
Colony Formation Assay for Hep3B and Huh7 Cells
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Hep3B and Huh7 cells (3 × 103/well) were seeded onto six-well plates and incubated at 37 °C. After 24 h, the cells were exposed to the concentrations (0, 20, 40, 80 μM) of Pyrogallol for 24 h and culture medium was replaced by a fresh one. After 10–12 days, cells were washed PBS, fixed, and stained with Diff quick solution (Sysmex, Kobe, Japan). The plate was dried at room temperature and colonies were manually counted under light microscopy.
3
Transwell Migration and Invasion Assay
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Transwell chambers uncoated (for migration assay) or coated (for invasion assay) with Matrigel (Corning Incorporated, Corning, NY, USA) were used according to the manufacturer's protocol. To measure the migration and invasive ability of cells, transfected cells were seeded into each upper chamber in serum-free media, and the lower chamber was filled with a complete medium supplemented with 5% or 10% FBS-containing media. The inserts of the invasion chamber were used after rehydration in a serum-free medium for 2 h before the cells were seeded. After 24 or 48 h of incubation, non-migrating or non-invading cells that remained on the top of the transwell insert were removed using a cotton swab. The migratory and invasive cells were then stained with Diff-Quick solution (Sysmex, Japan) and counted.
4
Morusin's Impact on Colony Formation
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HCT116 and SW480 cells (3 × 103/well) exposed to Morusin (0, 2.5, and 5 μM) for 24 h were distributed onto 6-well plates for a week. The cells were washed PBS, fixed, and stained with Diff quick solution (Sysmex, Kobe, Japan). Then, the colonies were counted under inverted microscope.
5
Invasion Assay Protocol for Cell Lines
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The cells were harvested and washed with PBS. After washing, 3x105 cells were resuspended in 500 µl serum-free medium and treated with different concentrations of DHMEQ. Cells were then transferred to the top of Matrigel-coated invasion chambers (24-well insert; Corning Inc.), and 750 µl of 20% FBS-RPMI-1640 was added to the bottom chamber. After 24 h of incubation, the non-invading cells were removed, and the invading cells that were attached to the bottom were fixed with methanol for 10 min and stained with Diff-Quick solution (Sysmex). Invading cells were photographed under the microscope at x10 magnification and counted.
6
Transwell Invasion Assay for Cell Motility
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Cell invasion ability was measured using the Transwell chamber system. Briefly, cultured cells were seeded onto the top of a 24-well Transwell filter chamber coated with 1 μg/mL Matrigel. A cell culture medium containing 5% bovine serum albumin and 20 μg/mL of fibronectin (Calbiochem, La Jolla, CA) was added to the bottom chamber. After 24 h of incubation, the cells were stained with a Diff-Quick solution (Sysmex, Kobe, Japan). The number or area of invaded cells was calculated using ImageJ (National Institutes of Health).
7
Sphere-forming and Soft-agar Clonogenic Assays
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For the sphere-forming assay, a single-cell suspension prepared by trypsinization was seeded in serum-free DMEM/Ham's F12 with EGF and bFGF (10 ng/ml each) at a density of 1×103 cells/ml. After 10 days of culture, spheres were attached to the plate by adding FBS (10%), stained using Diff-Quick solution (Sysmex Corporation, Kobe, Japan) and counted by eye. For the soft-agar clonogenic assay, cells were resuspended in the same volume of 0.7% agar with 10% FBS and plated onto the bottom agar layer (1:1 mixture of 1% agar and 2X DMEM/Ham's F12 with 10% FBS). After 14 days of incubation, colonies in five random fields/well were counted under a brightfield microscope (magnification, ×100).
8
Transwell Assay for Cell Invasion
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ES-2 cells were suspended in 500 μl of serum-free medium containing migracin A or the DMSO and seeded into the upper chambers coated with BD Matrigel Basement Membrane Matrix (Corning Inc., Corning, NY). The lower chambers were filled with 750μl of medium containing 10% FBS and incubated for 24 h at 37°C in a humidified CO2 incubator. Then, after fixation of the invading cells, non-invading cells remaining on the upper surface were removed by wiping with a cotton swab. Invading cells attached to the underside were stained with Diff-Quick solution (Sysmex, Kobe, Japan), and counted.
9
Differential Cell Counting in BALF
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Red blood cells (RBCs) in the precipitated cells obtained from the BALF samples as described above were removed using RBC Lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The total cells were counted using Nucleo Counter (ChemoMetec, Allerød, Denmark), and 10,000 cells from each sample were spun onto glass slides by cytocentrifugation (Cellspin, Hanil, Kimpo, South Korea) and stained with Diff-Quick solution (Sysmex Corporation, Hyogo, Japan). The number of eosinophils, macrophages, monocytes, lymphocytes, and neutrophils was determined by counting at least 200 cells in each of four different locations of each slide using a microscope (AXIO Imager 2; Carl Zeiss, Oberkochen, Germany).
10
Evaluating Invasive Potential of Cancer Cells
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The invasive potential of the cells was evaluated using the BioCoat Matrigel Invasion Chamber kit (BD Biosciences). Briefly, HSC‐4 cells were added to the Transwell insert chamber containing a filter coated with Matrigel at a density of 1.5 × 102 cells/μL. In the lower compartment, 750 μL of DMEM containing 10% FBS was used as the chemoattractant. The HSC‐4 cells were incubated with HSC‐4‐derived exosomes with or without 10 µmol/L of erlotinib or 100 µg/mL of cetuximab for 24 hours at 37°C. After removing the inserts, non–invading cancer cells remaining on the upper side of the filter were scraped off. Cells that invaded the lower side of the filter were then stained with the Diff‐Quick solution (Sysmex Corporation) at room temperature for 10 minutes, observed under a light microscope, and counted over five randomly selected fields at 200× magnification. Each experiment was performed in triplicate.
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