Immediately after assessment of airway hyperresponsiveness, mice were euthanized with a cocktail of ketamine (300 mg/kg) and xylazine (30 mg/kg) (Sigma Aldrich, St. Louis, MO) in an approximate volume of 100 μL per i.p. injection. The lungs were dissected and tracheostomized with an 18G luer stub adapter. The left lung was clamped using a hemostat and the right lung perfused with 500 μL sterile saline. Broncho-alveolar lavage fluid (BALF) was then pooled and aspirated into tubes.23 (link), 47 BALF was spun onto positively charged slides using Thermo Shandon Cytopsin® 3 (Marshal Scientific, Brentwood, NH). Slides were stained with Wright Geimsa stain (Sigma-Aldrich, St. Louis, MO) and washed with ddH20 for 10 minutes. The slides were cover slipped with Permount™ mounting media (Fisher Scientific, Pittsburgh, PA) and the number of eosinophils counted using the Labomed™ TCM 400 inverted microscope light microscope (Labo America Inc., Freemont, CA) in four randomly chosen fields of view at 20X. Eosinophils were determined as a percentage of the total cells in each field and expressed as percent eosinophil.23 (link), 47
Wright geimsa stain
Manufactured by Merck Group
Sourced in Germany
Wright Giemsa stain is a laboratory staining procedure used to visualize and differentiate cellular components, particularly in blood smears. It is a combination of two stains, Wright stain and Giemsa stain, which together provide a distinctive coloring pattern for various blood cells and cellular structures. The stain is commonly used in hematology and other clinical pathology applications to aid in the identification and analysis of blood cells.
Automatically generated - may contain errors
Lab products found in correlation
Ketamine, by Merck Group (1 mentions)
Xylazine, by Merck Group (1 mentions)
Permount mounting media, by Thermo Fisher Scientific (1 mentions)
Haemocytometer, by Nikon (1 mentions)
40 μm nylon mesh, by Thermo Fisher Scientific (1 mentions)
Percoll, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Dispase 2, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
2 protocols using wright geimsa stain
1
Murine Airway Inflammation Quantification
2
Isolation and Characterization of Murine Cell Populations
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Lung and liver lobes were cut by scissor into smaller fragments and suspended in media containing tissue digestion enzymes; DNAse I 5mg/mL, Collagenase D 30mg/mL and Dispase II 5mg/mL (all from Roche, Switzerland). Tissue was incubated in digestion buffer in a water bath with shaking at 37°C for 30 minutes. After, liver cells were resuspended in 36% Percoll (Sigma-Aldrich) (adapted from [17 (link)]) and centrifuged for 10mins at 500g without brake to separate leukocytes from fibrous tissue. Spleen was cut into smaller fragments but not subjected to enzymatic digestion. All tissues were strained using a 40μM nylon mesh (Fisher Scientific, USA). Red blood cells were lysed using Ammonium-Chloride-Potassium buffer as previously described [18 (link)]. Cell pellets were resuspended and viable cells counted using Trypan blue staining solution on a haemocytometer (Nikon, Tokyo Japan). Cytospins were performed on glass covered slips stained with Wright-Geimsa stain (Sigma-Aldrich) and imaged using a light microscope (Leica, Germany).
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