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Diff quick dye

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Diff-Quick dye is a three-step staining method used for the rapid differential staining of blood smears and other cytological preparations. It is a reliable and efficient tool for the identification and differentiation of various cell types, such as red blood cells, white blood cells, and platelets. The Diff-Quick dye provides a quick and easy way to obtain high-quality staining results for microscopic analysis.

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Lab products found in correlation

Cell invasion assay kit, by Merck Group (1 mentions) Transwell insert, by BD (1 mentions) Matrigel invasion chamber, by BD (1 mentions) 24 well insert, by BD (1 mentions)

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Diff-Quick

6 protocols using diff quick dye

1

Quantification of Intra-Alveolar PMNs

Cited 1 time
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At the end of the experiments, BAL was performed to enumerate the absolute number of PMNs in the intra-alveolar inflammatory exudates as described previously (27 (link)). In brief, lungs were lavaged by an intratracheal injection of 1 ml PBS followed by gentle aspiration. The procedure was repeated three times. After the collected BAL fluid was centrifuged, cell pellets were suspended in PBS. Cell suspension (300 μl) was cytocentrifuged on to a glass slide at 300 rpm for 5 min with a cytocentrifuge (Shandon). Slides were stained with Diff-Quick dye (Dade Behring) and examined at magnifications of 20× and 40× by light microscopy. The percentage of PMNs was calculated after counting 300 cells in randomly selected fields.
Jiang C., Liu Z., Hu R., Bo L., Minshall R.D., Malik A.B, & Hu G. (2017). Inactivation of Rab11a GTPase in macrophages facilitates phagocytosis of apoptotic neutrophils. Journal of immunology (Baltimore, Md. : 1950), 198(4), 1660-1672.
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2

Quantification of Neutrophils in BAL

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At the end of the experiments, the absolute number of PMNs in BAL fluid was counted as described previously [27 (link)]. In brief, the collected BAL fluid was centrifuged and cell pellets were suspended in PBS. Cell suspension (300 μl) was cytocentrifuged onto a glass slide with a cytocentrifuge (Shandon, Southern Sewickley, PA, USA). Slides were stained with Diff-Quick dye (Dade Behring, Newark, DE, USA) and analyzed by light microscopy (magnification, 20× and 40×). The percentage of PMNs was calculated after counting at least 300 cells in five or more randomly selected fields in each slide.
Du X., Jiang C., Lv Y., Dull R.O., Zhao Y.Y., Schwartz D.E, & Hu G. (2017). Isoflurane promotes phagocytosis of apoptotic neutrophils through AMPK-mediated ADAM17/Mer signaling. PLoS ONE, 12(7), e0180213.
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3

Transwell Invasion Assay for Folate-Dependent Cell Lines

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Invasion assays were carried out using inserts precoated with extracellular matrix, and the Cell Invasion Assay Kit (Chemicon) according to the manufacturer’s protocol. Cells were kept in standard or folate-free medium for 72 h and then seeded into the upper chamber of a Transwell insert (BD Bioscience) in serum-free medium at a density of 5×104 cells/well. Medium containing 10% FBS was placed in the lower chamber as a chemoattractant, and cells were incubated for 24 h at 37 °C in CO2 incubator. Nonmigrating cells were removed from the upper chamber by scraping; the remaining cells were stained with Diff-Quick dye (Dade Behring, Inc.). Stained cells were counted in 10 random microscopic fields in three independent inserts. Alternatively, stained cells were lysed with 10% acetic acid (100–200 μl/well) and quantified by absorbance at 560 nm. In experiments with replete medium, cells were grown in folate-free medium for 72 h and then in medium with 2.2 μM folic acid for 72 h.
Ashkavand Z., O’Flanagan C., Hennig M., Du X., Hursting S.D, & Krupenko S.A. (2017). Metabolic reprogramming by folate restriction leads to a less aggressive cancer phenotype. Molecular cancer research : MCR, 15(2), 189-200.
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4

Fibronectin-Mediated Cell Migration and Invasion Assay

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Cells were seeded into the upper chamber of a Transwell insert pre-coated with 5μg/mL fibronectin for migration or a BD Matrigel invasion chamber for invasion in serum-free medium at a density of 50,000 cells per well (24-well insert; 8μm pore size, BD Biosciences, San Jose, CA). A medium containing 10% fetal bovine serum (FBS) was placed in the lower chamber to act as a chemoattractant, and cells were further incubated for the indicated time points. Non-migratory cells were removed from the upper chamber. The migratory cells remaining on the lower surface of the insert were stained using Diff-Quick dye (Dade Behring, Inc, Newark, DE). Cells were quantified as the average number of cells found in five random microscope fields in three independent inserts.
Findlay V.J., Wang C., Nogueira L.M., Hurst K., Quirk D., Ethier S.P., Staveley O'Carroll K.F., Watson D.K, & Camp E.R. (2014). SNAI2 modulates colorectal cancer 5-fluorouracil sensitivity through miR145 repression. Molecular cancer therapeutics, 13(11), 2713-2726.
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5

Quantifying Alveolar Macrophage Phagocytosis

Cited 2 times
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The lungs were aspirated 3 times with 1 ml of sterile PBS per mouse to collect bronchoalveolar lavage (BAL) fluid. The BAL fluid was pooled and centrifuged, and cell pellets were suspended in PBS. Cell suspensions were cytospun onto slides with a cytocentrifuge (Shandon, Southern Sewickley, PA, USA). Slides were stained with Diff-Quick dye (Dade Behring, Newark, DE, USA) and examined at magnifications of 20× and 40× by light microscopy. At least 300 macrophages were counted in each sample. Phagocytosis was expressed as the percentage of alveolar macrophages containing apoptotic bodies [24 (link)].
To assess the alveolar macrophages’ ability to remove apoptotic PMNs in vivo, apoptotic mouse PMNs (1.0 × 107 in 100 μl) were labeled with pHrodo™ Red (SE) (Invitrogen, Carlsbad, CA, USA) and intratracheally instilled into mice [25 (link)]. The fluorescence of pHrodo™ Red dramatically increases in phagosomes of macrophages as pH decreases from neutral to acidic. Attached apoptotic PMNs were excluded because pHrodo-SE was nonfluorescent at neutral pH. After 3 hours of PMN instillation, BAL was performed. Cells in BAL fluid were washed, resuspended and analyzed by flow cytometry.
Du X., Jiang C., Lv Y., Dull R.O., Zhao Y.Y., Schwartz D.E, & Hu G. (2017). Isoflurane promotes phagocytosis of apoptotic neutrophils through AMPK-mediated ADAM17/Mer signaling. PLoS ONE, 12(7), e0180213.
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6

Bronchoalveolar Lavage Cell Analysis

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At the end of the experiments, BAL was performed by intratracheal injection of 1
ml PBS followed by gentle aspiration. The lavage was repeated three times. The pooled BAL
fluid was centrifuged, and cell pellets were suspended in PBS. Cell suspensions were
cytospun onto slides with a cytocentrifuge (Shandon, Southern Sewickley, PA). Slides were
stained with Diff-Quick dye (Dade Behring, Newark, DE) and examined at a magnification of
×20 and ×40 by light microscopy. The percentage of PMNs was determined
after counting 300 cells in randomly selected fields.
Yang Z., Sun D., Yan Z., Reynolds A.B., Christman J.W., Minshall R.D., Malik A.B., Zhang Y, & Hu G. (2014). Differential role for p120-catenin in regulation of TLR4 signaling in macrophages. Journal of immunology (Baltimore, Md. : 1950), 193(4), 1931-1941.
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