Macrophages were also cultured for 7 days with the cytokines in chamber slides as described before. The slides were rapidly dried well in the air, and then fixed by May-Grunwald solution (Muto Pure Chemicals, Tokyo, Japan) for 2 min at RT. Equal volume of phosphate buffer pH 6.4 (Muto Pure Chemicals) was applied on the slides with May-Grunwald solution for 2 min at RT. After a 20-s rinse with Giemsa solution (Muto Pure Chemicals) diluted by phosphate buffer pH 6.4, the slides were incubated with Giemsa solution for 15 min at RT. Following short rinse by distilled water, the samples were rapidly dried well in the air, immersed in xylene, and mounted with the non-aqueous mounting medium DPX (Merck, Darmstadt, Germany).
Giemsa solution
Manufactured by Muto Pure Chemicals
Sourced in Japan
Giemsa solution is a staining reagent commonly used in microscopy and hematology. It is a polychromatic stain that selectively colors various cellular components, enabling the differentiation and identification of different cell types. The solution contains a mixture of methylene blue, eosin, and azure dyes that provide distinct staining patterns for nuclei, cytoplasm, and other cellular structures.
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Lab products found in correlation
Fibronectin, by Merck Group (2 mentions)
Cell culture insert with 8.0 μm pore size polyester filters, by BD (2 mentions)
Giemsa solution, by Olympus (1 mentions)
Cytospin 4 cytocentrifuge, by Thermo Fisher Scientific (1 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Glass coverslip, by Matsunami (1 mentions)
Mgk s mounting solution, by Matsunami (1 mentions)
May grunwald solution, by Muto Pure Chemicals (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
8 protocols using giemsa solution
1
Macrophage Culture and Staining Protocol
2
Cell Migration Assay Using Fibronectin
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The cell migration assay was carried out using the Cell Culture Insert with an 8.0‐μm pore size PET filter (BD Biosciences, Billerica, MA, USA). Before the assay, the lower surface of the filter was immersed for 30 min in 10 μg/mL fibronectin (Sigma) diluted with PBS. RPMI‐1640 medium containing 10% FBS was added to the lower chamber. Subsequently, the same number of cells per well were suspended in RPMI‐1640 medium containing 10% FBS and added to the upper chamber. After incubation for 24 h at 37°C in a humid 5% CO2 atmosphere, the cells on the upper surface of the filter were completely removed by wiping with cotton swabs. The cells on the lower surface of the filter were fixed in methanol for 30 min, washed with PBS, and then incubated with Giemsa solution (Muto Pure Chemicals, Tokyo, Japan) for 30 s. The cells on the lower surface were counted in at least five fields at a magnification of ×200 under a microscope.
3
Migration Assay Using Cell Inserts
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Migration assay was done by using a cell culture insert with 8.0‐μm pore size polyester filters (BD Biosciences). Before the assay, the lower surface of the filter was immersed for 30 minutes in 10 μg/mL fibronectin (Sigma) diluted with PBS. Then, we added RPMI‐1640 medium with 10% FBS in the lower chamber and RPMI‐1640 medium with 10% FBS containing cells (5 × 103) in the upper chamber. After 48 hours of incubation, we analyzed cells on the lower side of the inserts. We fixed cells in methanol for 30 minutes. Fixed cells were washed with PBS and stained with Giemsa solution (Muto Pure Chemicals) for 30 seconds. Cells that migrated to the lower surface were counted in five randomly selected fields under a microscope at a magnification of ×200.
4
Giemsa Staining of Cells
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Giemsa solution (Muto Pure Chemicals) was diluted 20 times by PBS (Muto Pure Chemicals) of pH 6.4 and a final concentration of 1/150 M. The cells were cytospun onto glass slides and fixed with methanol for 2 min. The cells were stained with the Giemsa solution for 20 min at room temperature, washed, and analyzed by microscopy (BX53; Olympus).
5
Cell Migration Assay with Transfected Cells
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Migration assays were performed by using the Cell Culture Insert with 8.0-μm pore size polyester filters (BD Biosciences). We added 700 μL of cultured medium for PC3 or DU145 cells in the lower chamber and 300 μL of cultured medium containing cells transfected with siRNAs (5 × 103) in the upper chamber. After 24 h of incubation, the cells on the lower side of inserts were fixed, then stained with Giemsa solution (Muto Pure Chemicals, Tokyo, Japan). The cells that migrated on the lower surface were counted in four randomly selected fields under a microscope at a magnification of × 40.
6
Cell Migration Assay Protocol
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The cell migration assay was performed as previously described.26 (link) Briefly, 50 000 cells were suspended in 30 μL of RPMI 1640 (LNCaP cells) or DMEM (PC3 and VCaP cells) medium containing 10% FBS and added to the upper chamber. After incubation for 24 h in LNCaP and PC3 cells or 48 h in VCaP cells at 37°C in a humid 5% CO2 atmosphere, the cells on the lower surface of the filter were fixed in methanol for 30 min, then stained with Giemsa solution (Muto Pure Chemicals, Tokyo, Japan) for 30 s. The cells on the lower surface were counted in at least five fields at a magnification of ×200 under a microscope.
7
Tumor Cellularity Assessment Protocol
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To assess tumor cellularity, all samples were stained with Cyto Quick A solution (Muto Pure Chemicals, Tokyo, Japan) for 5 sec, then stained with Cyto Quick B solution for 15 sec. TIC samples were stained with Pap or Giemsa stain. Pap staining was performed by placing samples in the following solutions in order: 70% ethanol for 30 sec, water for 50 sec, hematoxylin for 2 min, water for 1 min, 70% ethanol for 30 sec, 0.3% hydrochloric acid/70% ethanol for 15 sec, water for 3 min, 70% ethanol for 30 sec, 95% ethanol for 30 sec ×2, OG6 solution (Muto Pure Chemicals) for 2 min, 95% ethanol for 30 sec ×3, EA50 solution (Muto Pure Chemicals) for 3 min, 95% ethanol for 30 sec ×2, 100% ethanol for 30 sec ×3, and xylene for 30 sec ×3. Giemsa staining was performed using the following solutions: May‐Grünwald solution (Muto Pure Chemicals) for 3 min, water for 1 min, Giemsa solution (Muto Pure Chemicals) for 15 min, and water for 1 min. Slides were stored at 4°C until required for DNA extraction.
8
Giemsa Staining of Cytospin Slides
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