For phenotypic characterization, differentiated hCD34+-derived cells were cytocentrifuged on a Cellspin II with an EASY rotor (Tharmac/Hettich) and sequentially stained in dianisidine (1.5% o-Dianisidine; Sigma–Aldrich; in methanol) for 2 min, in H2O2/ethanol solution (50% ethanol, 0.9% H2O2 in distilled water) for an additional 2 min, in May–Grünwald staining solution (Sigma–Aldrich) for 5 min, and finally in Giemsa staining solution (Fluka Analytica; Sigma–Aldrich) for 10 min. After a rinse in distilled water, sample slides were air-dried and mounted (Entellan®; Merck) with a coverslip. Slides were visualized under an IX73P1F inverted microscope, using LED illumination, a 40 × lens, an XC50 camera, and averaging seven frames per image in CellSens 1.7 (Olympus Corp.).
May gr nwald staining solution
Manufactured by Merck Group
May–Grünwald staining solution is a laboratory reagent used for staining biological samples. It is commonly used in cytology and histology for the differentiation and visualization of cellular components.
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Lab products found in correlation
O dianisidine, by Merck Group (1 mentions)
Cellsens 1, by Olympus (1 mentions)
Entellan, by Merck Group (1 mentions)
Evos xl core cell imaging system, by Thermo Fisher Scientific (1 mentions)
Giemsa staining solution, by Merck Group (1 mentions)
G csf, by R&D Systems (1 mentions)
Pe conjugated rat anti mouse cd11b, by BD (1 mentions)
Dnp bsa, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
H dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Te2000 s microscope, by Nikon (1 mentions)
Shandon cytospin, by Thermo Fisher Scientific (1 mentions)
Giemsa stain, by Merck Group (1 mentions)
Kx 21n, by Sysmex (1 mentions)
4 protocols using may gr nwald staining solution
1
Phenotypic Characterization of Differentiated hCD34+ Cells
2
Monocyte Differentiation Assay
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Cells were washed with PBS twice and inoculated into 24-well plates at 1 x 105 cells/ml in the culture medium with 1 ng/ml IL-3, 10 ng/ml G-CSF (R&D Systems, Minneapolis, MN), 1 μg/ml FL-BSA, or 0.1 μg/ml DNP-BSA (Thermo Fisher Scientific). On day 5 or 6, the cells were stained with 50 μl PBS containing PE-conjugated rat anti-mouse CD11b (1:100 dilution; BD Biosciences) or PE rat IgG2b κ isotype control (1:100 dilution; BD Biosciences). The cells were incubated on ice for 30 min, washed twice with PBS, and resuspended with 200 μl PBS. The cells were analyzed by the FACSCalibur flow cytometer.
For May-Grünwald-Giemsa staining, the cells cultured in each ligand for 5 days were washed by PBS once and fixed on glass slides with methanol for 90 sec. The cells on the glass slides were immersed in May-Grünwald staining solution (Sigma-Aldrich) for 5 min, immersed in PBS for 3 min, immersed in Giemsa staining solution (Sigma-Aldrich) for 15 min, and rinsed in Milli-Q water twice. The stained cells were photographed with EVOS XL Core Cell Imaging System (Thermo Fisher Scientific).
For May-Grünwald-Giemsa staining, the cells cultured in each ligand for 5 days were washed by PBS once and fixed on glass slides with methanol for 90 sec. The cells on the glass slides were immersed in May-Grünwald staining solution (Sigma-Aldrich) for 5 min, immersed in PBS for 3 min, immersed in Giemsa staining solution (Sigma-Aldrich) for 15 min, and rinsed in Milli-Q water twice. The stained cells were photographed with EVOS XL Core Cell Imaging System (Thermo Fisher Scientific).
3
Quantitative Analysis of C2C12 Myotube Formation
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C2C12 cells, a mouse myoblast cell line, were used for in vitro experiments. C2C12 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium‐high glucose medium (DMEM‐h; #11960044, Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) foetal bovine serum (FBS; #16000, Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (P/S; #15140, Gibco). Cells were subcultured at intervals of 3–4 days and used for the experiment. For myoblast differentiation, cells were cultured in a differentiation medium supplemented with 2% horse serum (Gibco) and 1% glutamine (Gibco) for 5–7 days. May–Grünwald and Giemsa staining were performed for quantitative measurement of myotubes. Differentiated C2C12 cells were washed with cold phosphate‐buffered saline (PBS) and fixed with 100% methanol. Subsequently, the May–Grünwald staining solution (Sigma) diluted with sodium phosphate buffer (pH 6.0) was added to the cells for 5 min. Cells were then washed with distilled water and incubated for 10 min in Giemsa staining solution. After washing twice with distilled water, the diameter of myotubes was quantified using an inverted microscope (Nikon, Japan).
4
Automated Blood Analysis of Diseased Mice
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Diseased mice were subjected to automated blood counting (Sysmex KX-21N) analyses. Morphological analysis of GFP+ BM cells were spun on a glass slide using a Shandon Cytospin (Thermo Scientific) and air-dried. Slides were stained for 5 min in May-Grünwald staining solution (Sigma-Aldrich), washed for 5 min in phosphate-buffered saline, and stained for 17 min in Giemsa stain (Sigma-Aldrich) diluted 1:20 in deionized water. Slides were rinsed in deionized water, air-dried, and visualized under a Nikon TE2000-S microscope. For each sample, four to five images were taken by a QImaging camera and QCapture Pro software (Fryer Company Inc.). Two hundred cells from each sample were evaluated to quantify blasts and differentiated cells.
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