Hema 3 stain kit

Granulocytes were isolated from peripheral blood of allergic or healthy donors. Eosinophils were enriched and purified by negative selection using the human eosinophil enrichment cocktail (SSep™, StemCell Technologies, Seattle, WA, USA) and the MACS bead procedure (Miltenyi Biotec, Auburn, CA, USA), as previously described (23 (link)) with the exception that hypotonic red blood cell (RBC) lysis was omitted to avoid any potential for RBC lysis to affect eosinophil function. Eosinophil viability and purity were greater than 99% as determined by ethidium bromide (Molecular Probes, Life Technologies, Carlsbad, CA, USA) incorporation and cytocentrifuged smears stained with HEMA 3 stain kit (Fisher Scientific, Medford, MA, USA), respectively. Purified eosinophils (10⁶ cells/mL) were stimulated with TNF-α (200 ng/mL; R&D Systems, Minneapolis, MN, USA) or recombinant human CCL11 (100 ng/mL; R&D Systems), in RPMI-1640 medium plus 0.1% ovalbumin (Sigma, St. Louis, MO, USA), or medium alone at 37°C, for 1 h. At these concentrations, CCL11 and TNF-α induce consistent cell secretion (16 (link)).

The lung of each mouse was lavaged with 3 × 0.5 ml of sterile saline according to previously described^{59 (link),60 (link)}. The BALF was centrifuged at 250 × g for 10 min. The pellet was resuspended with 1 ml of sterile saline, and then the cells were mounted on a slide by cytospin centrifuge (Hettich ROTOFIX 32 A) at 1000 rpm for 3 min. Slides were air-dried and stained with Hema-3 Stain Kit (Fisher Scientific Company, Kalamazoo, MI) for analysis with Nikon Eclipse TE2000-U research microscope (Nikon, Melville, NY).

Blood was collected from individual fish by venipuncture of the caudal vein using a 21 gauge needle and tuberculin syringe. The needle was removed and blood gently expressed into a 400 μl heparinized Microtainer tube (Becton-Dickinson). Blood smears were prepared by spreading a drop of blood onto the surface of an RNAse free MMI membrane slide and support (Molecular Machines & Industries). Smears were allowed to air dry and stained using a Hema-3 stain kit (Fisherbrand). To prevent cross-contamination, smears were stained by individually flooding slide surfaces, each with fresh reagents, rather than by dipping them into a common staining jar. Slides were examined at 400 X using an Olympus IX71 microscope and erythrocytes excised using an MMI CellCut Plus laser microdissection and isolation cap system (Molecular Machines & Industries). Only widely separated erythrocytes were excised near the feather edge of the smear. Approximately 100–150 erythrocytes were collected per isolation tube. DNA was isolated from erythrocytes using DNeasy mini spin columns per manufactures instruction (Qiagen DNeasy).

To determine the bacterial burden in an individual epithelial cell, we utilized an invasion index to represent the proportion of infected cells and how heavily each cell was colonized [40 (link),41 (link)].To calculate the invasion index in epithelial cells, tear fluid was collected from the ocular surface bacterial inoculation in the absence or presence of 10 nM of E₆₄, for 6 h, the cells was sedimented by centrifugation at 4°C at 250xg for 10 min and washed three times with cold PBS followed by sedimentation. The final suspension was resuspended in 200 μl of PBS and the cells were mounted on slides by cytospin centrifugation. The slides were stained using the Hema-3 Stain Kit (Fisher Scientific, Pittsburgh, PA). With light microscopy, one hundred randomly selected epithelia per slide were analyzed at ×1,000 magnification. Bacteria at adherence and internalization steps were counted as invasion bacteria [35 (link)]. The invasion index was calculated and expressed as the ratio of invasion bacteria to total bacterium-laden epithelial cells. The percentage of epithelial cells invaded by S. aureus was also calculated.

Granulocytes were isolated from peripheral blood of allergic or healthy donors. Eosinophils were enriched and purified by negative selection as previously described (StemSep^TM, StemCell Technologies, Seattle WA; Miltenyi Biotec, Auburn, CA; Bandeira-Melo et al., 2000 (link); Akuthota et al., 2014 (link)). The hypotonic red blood cell (RBC) lysis was omitted to avoid any potential for RBC lysis to affect eosinophil function. Eosinophil viability and purity were >99% as determined by ethidium bromide (Molecular Probes, Life Technologies, Carlsbad, CA) incorporation and cytocentrifuged smears stained with HEMA 3 stain kit (Fisher Scientific, Medford, MA), respectively. Purified eosinophils (10⁶ cells/mL) were stimulated with TNF-α (200 ng/mL; R&D Systems, Minneapolis, MN) or recombinant human CCL11 (100 ng/mL; R&D Systems), in RPMI-1640 medium plus 0.1% ovalbumin (OVA; Sigma, St. Louis, MO, USA), or medium alone at 37°C, for 1 h as before (Carmo et al., 2016 (link)).

To qualitatively evaluate in vivo uptake of each particle type by alveolar macrophages and approximate intracellular location, phase contrast imaging of fixed cells from bronchoalveolar lavage (BAL) was conducted (n = 8) as previously described [17 (link)]. Briefly, a cannula was inserted into the esophagus of euthanized mice, attached to a sterile syringe, then followed by the addition 0.8 ml of ice cold phosphate-buffered saline to the entire lung. Mild compressions of the chest were performed as fluid was collected into the syringe. Lavage was performed 4 times and centrifuged to pellet collected cells. BAL cells were resuspended in saline and counted. Collected BAL cells were immobilized via Cytospin, fixed and stained with a HEMA3 stain kit (Fisher Scientific), and imaged with a Leica DM2500 equipped with an Olympus DP73 digital camera. Images were evaluated for intracellular and extracellular particulate and compared to both in vitro TEM images and in vivo stained histological sections of lung.