Heparinized venous blood was obtained from both atopic and non-atopic volunteers (>6 years of age) in accordance with a protocol approved by the Institutional Review Board for Human Subjects at CCHMC (IRB 2008–0090). Granulocytes were isolated using dextran sedimentation followed by density gradient separation as described38 (link). Following isolation of the granulocyte layer, purified eosinophils were collected by negative selection using the eosinophil-isolation kit (#130–042-901, Mitenyi Biotec). Eosinophil and neutrophil purity was assessed by HEMA-3 staining (23–123869, Fisher Scientific) followed by microscopic analysis, and the suspensions were routinely 95–98% pure for the respective granulocyte.
Hema 3 staining
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HEMA-3 staining system is a laboratory equipment used for the rapid staining of blood smears and other cytological preparations. It provides a quick and reliable method for differentiating and identifying various types of blood cells.
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Lab products found in correlation
5 protocols using hema 3 staining
1
Isolation of Eosinophils and Neutrophils
2
Bronchoalveolar Lavage Neutrophil Quantification
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The detailed procedures were described previously [28 (link), 29 (link)]. The mice were intubated with a 24-gauge cannula after their tracheas were surgically isolated. The lungs were flushed with 1 ml PBS back and forth for 3 times. Protein concentration in the BAL was determined by a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). BAL cell smear was made using cytospin (Shandon, Pittsburgh, PA, USA). The slides were visualized using Hema3 staining (Fisher Scientific, Middletown, VA, USA). Neutrophils and monocytes were identified by a certified laboratory technologist in a blinded fashion. BAL cells were also used for flow cytometry analysis after lysing erythrocytes.
3
Isolation of Eosinophils and Neutrophils
4
Quantifying Inflammatory Response in Mice
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The infiltration level of polymorphonuclear neutrophils (PMNs) in the serum was used to assess the degree of inflammatory response as previously described (33 (link)). The animal blood was obtained by cardiac blood sampling on the sacrificed mouse and was stained on the smear by HEMA-3 staining (23-122929, Thermo Fisher), quantified by light microscopy at 400 × final magnification.
5
Transwell Invasion Assay for Metastatic Cells
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The migratory capacity of metastatic cells was measured by their ability to invade through extracellular matrix (ECM), following a previously described protocol (12 (link)). Cells (~5,000) were plated in triplicates in 8-µm pore-sized cell culture inserts (BD Labware; BD Biosciences) and supplemented with media without serum. These cell inserts were positioned in 12-well plates containing media with 10% FBS, followed by incubation for 12–16 h at 37°C in a 5% CO2 incubator. Migration of cells was visualized by staining the cells with Hema 3 staining following manufacturer's instructions (Thermo Fisher Scientific, Inc.) and images were captured at 100× magnification using a microscope (Nikon Eclipse TE2000; Nikon Corporation). Total number of stained and migrated cells were counted, and calculated as relative migration units between CRC cells.
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