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Shandon kwik diff

Manufactured by Thermo Fisher Scientific

The Shandon Kwik-Diff is a rapid staining system designed for the preparation of blood smears and other cytological samples. It provides a fast and consistent staining process for the identification of various cell types and structures.

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3 protocols using shandon kwik diff

1

Tracheal Aspirate Sampling for Gene Expression

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Tracheal aspirate samples were collected within 72 hours of endotracheal intubation with an inline Ballard suction catheter (Halyard, Alpharetta, GA) connected to a sterile Lukens trap (Covidien, Walpole, MA) using up to 5 mL of sterile saline and processed according to published protocols (19 (link), 20 (link)). For infants and toddlers, 1–2 mL of saline is instilled. The Ballard inline suction catheter is passed once to obtain a sample. If 50% of the instilled volume is not returned, then a repeat passage of the Ballard is performed. The Ballard suction catheter (Halyard, Alpharetta, GA) may be flushed with additional saline to move the aspirate into the Lukens trap (Covidien, Walpole, MA). Cell viability was determined with trypan blue exclusion on a Countess hemocytometer. Cell purity was assessed by Shandon Kwik-Diff (Thermo Scientific, Waltham, MA) staining of cytospin cell preparations (20 (link)). Up to 1 × 106 cells were stored in RNALater at –80°C until RNA was extracted for gene expression analysis.
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2

Quantifying Airway Inflammation via BALF

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Bronchoalveolar lavage fluid (BALF) was extracted to determine levels of inflammation within the airways. Cold PBS was flushed into the lungs of mice through a small incision in the trachea; briefly, a 19-gauge needle was inserted into the incision and 4 volumes of PBS (1x400 µL and 3x300 µL) flushed into the lungs. The number of live cells in the BALF collected was counted using a haemocytometer.
Fifty thousand cells were then centrifuged at 400 xg for 5 minutes onto a frosted slide using a Cytospin 3 (Shandon) for differential cell analysis. Slides were fixed with 2-propan-2-ol for 1 minute and left to dry overnight. Slides were then submerged in rapid 1 dye for 12 dips and then immediately placed into rapid II for 6 dips (Shandon Kwik Diff; Thermofisher Scientific). Slides were then left to dry before applying a small drop of DPX mounting media to the cytospot and applying a coverslip. Slides were then imaged under a microscope with 20x magnification. A total of 500 cells were counted from random fields, including macrophages, lymphocytes and neutrophils, based on standard morphological criteria.
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3

Liver and Lung Tissue Analysis

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Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined spectophotometrically using standard kits (Thermo Fisher Scientific, Waltham, MA).
Formalin fixed, paraffin-embedded liver and lung tissue was cut at 5 µm and mounted on glass slides, and stained with hematoxylin and eosin (H&E). Neutrophils were visualized by staining for chloroacetate esterase (CAE) by incubating tissue sections in a solution of napthol AS-D chloroacetate (1 mg/ml) in N,N-dimethylformamide, with 4% sodium nitrite and 4% new fuchsin. Tissue sections were visualized on a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan) with Metamorph software (Molecular Devices, Sunnyvale, CA).
Cells in BALF were counted using a hemocytometer, and cells were spun onto glass slides using a Cytospin centrifuge. Cells were stained with the Shandon Kwik-Diff (Thermo Fisher Scientific) differential staining kit according to manufacturer’s instructions. Slides were visualized on a Nikon Eclipse E600 microscope (Nikon Corporation) with Metamorph software (Molecular Devices), and total number of neutrophils per 200 total cells were counted using Image J software.
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