The cells (1×106) were seeded into a 6-well plate. After heat stress, the media was removed and the cells were fixed for 5 min in methanol, and then dried completely. Giemsa stain (Sigma, MA, USA) was diluted with deionized water (1:20), and the samples were stained with Giemsa stain for 30 min. The stain was washed with running tap water and air dried, and the images were observed using an inverter microscope (40×; CKX53, Olympus, Tokyo, Japan).
Giemsa stain
Manufactured by Merck Group
Sourced in United States, Germany, India, United Kingdom, Japan, Sao Tome and Principe, Macao, Nigeria, China, Czechia, Canada, Singapore
Giemsa stain is a biological stain used in microscopy. It is a mixture of methylene blue, eosin Y, and azure B that is commonly used to stain blood smears, bone marrow aspirates, and other cytological preparations. The stain allows for the visualization and differentiation of cellular components, such as nuclei, cytoplasm, and various organelles, in a variety of cell types.
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Lab products found in correlation
Methanol, by Merck Group (13 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Matrigel, by BD (8 mentions)
Digital camera, by Olympus (8 mentions)
May grunwald stain, by Merck Group (8 mentions)
Penicillin, by Merck Group (7 mentions)
Streptomycin, by Merck Group (7 mentions)
Fetal bovine serum (fbs), by Merck Group (6 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
May gr nwald, by Merck Group (5 mentions)
Cytochalasin b, by Merck Group (5 mentions)
Sodium chloride (nacl), by Merck Group (5 mentions)
Trypan blue, by Merck Group (4 mentions)
Crystal violet, by Merck Group (4 mentions)
Cytospin 4, by Thermo Fisher Scientific (4 mentions)
Potassium chloride (kcl), by Merck Group (4 mentions)
Entellan, by Merck Group (3 mentions)
May gr nwald stain, by Merck Group (3 mentions)
Chloroquine diphosphate, by Merck Group (3 mentions)
292 protocols using giemsa stain
1
Giemsa Staining of Heat-Stressed Cells
2
Cell Migration and Invasion Assays
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In the migration assay, SCC9 stable clones (3 × 104 cells) and TW2.6 stable clones (5 × 104 cells) were seeded in the upper chamber of 24-well Transwell inserts (Millipore, Bedford, MA, USA) in serum-free medium. After 24 h of incubation at 37 °C, filters were fixed with methanol and stained with Giemsa stain (Sigma). Migrated cells were counted under an inverted microscope in six randomly chosen fields.
For invasion assay, 60 μl Matrigel (25 mg/50 ml; BD Biosciences, MA, USA) was added to Transwell inserts. SCC9 stable clones (3 × 104 cells) and TW2.6 stable clones (5 × 104 cells) were seeded in the upper chamber of Matrigel-coated Transwell inserts in serum-free medium. After 48 h of incubation at 37 °C, filters were fixed with methanol and stained with Giemsa stain (Sigma). Invasive cells were counted under an inverted microscope in six randomly chosen fields.
For invasion assay, 60 μl Matrigel (25 mg/50 ml; BD Biosciences, MA, USA) was added to Transwell inserts. SCC9 stable clones (3 × 104 cells) and TW2.6 stable clones (5 × 104 cells) were seeded in the upper chamber of Matrigel-coated Transwell inserts in serum-free medium. After 48 h of incubation at 37 °C, filters were fixed with methanol and stained with Giemsa stain (Sigma). Invasive cells were counted under an inverted microscope in six randomly chosen fields.
3
Microscopic Detection of Malaria Parasites
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Blood smears (two thick and thin smears) were prepared at baseline, hour (H) 8, H16, H24, H32, H40, H48, H56, H64, H72, and at days 4, 5, 6, and 7. The smears were dried and stained with either 10% Giemsa stain (Cat: 1.09204, Sigma–Aldrich) for 15 min for screening smears or with 3% Giemsa stain for 60 min for all other timepoints. Each stained smear was read by Lambaréné method24 (link) by two independent microscopists qualified for the detection of asexual and sexual Plasmodium parasites. A smear was considered negative if no asexual parasites or gametocytes were detected in 200 high-power fields instead of the usual 100 fields. Asexual parasite density, expressed as parasites per μl (P/μl) of blood, was calculated by averaging the two counts. Microscopy results were considered non-concordant in case of difference of parasitaemia of ≥50% for parasitaemia ≥300P/μl, difference of parasitaemia of ≥100P/μl for parasitaemia <300P/μl, difference in positivity, or difference in Plasmodium species determination. Non-concordance was resolved by a third microscopist.
4
Malaria Diagnostic Microscopy Protocol
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For routine surveillance, thick and thin blood films were prepared using standard procedures. Briefly, for thin smears one drop of blood was spread to a monolayer onto a glass slide using the edge of another glass slide, quickly air dried, fixed with absolute methanol and stained 20 min by 10 % Giemsa stain (Merck, Singapore) in pH = 7.2 phosphate buffer; for thick smears one drop of blood was smeared onto a glass slide, slowly and fully dried at room temperature or in an incubator, de-haemoglobinized in water and stained 8 min by 10 % Giemsa stain (Merck, Singapore) in pH = 7.2 phosphate buffer. Then all the smears were protected by a cover slip mounted with Eukitt ® (Sigma-Aldrich, Singapore) mounting medium and dried before reading. Blood smears were examined under ×1000 magnification with an Olympus CX31 microscope (Olympus, Singapore) and microphotographs taken with a Nikon Eclipse 80i microscope equipped with a Nikon DS Ri1 camera and the Nikon NIS Elements D Imaging Software (Nikon, Singapore).
5
Hemolymph Collection and Staining
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The hemolymph was withdrawn from the heart using a capillary tube [39 (link)]. Part of the collected hemolymph was put on a glass slide, making a monolayer of hemocytes. Then the slides were dried in a moist chamber for 15 min at room temperature, followed by 5 min of dehydration in methanol, and finally stained for 20 min with 10% Giemsa stain (Aldrich) [40 (link)].
6
Hemolymph Sampling and Hemocyte Preparation
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Hemolymph samples were collected from each group of tested snails that was exposed to LC10, LC25, and control snails group by removing a small portion of the shell and inserting a capillary tube into the heart (Nduku and Harrison 1980 (link)). Hemocytes monolayers were prepared by placing 10 μl of hemolymph on a glass slide and were allowed to be adhered the glass surface for 15 min at room temperature. Hemocytes were fixed with absolute methanol for 5 min and then stained with 10% Giemsa stain (Aldrich) for 20 min (Abdul-Salam and Michelson 1980 (link)), then examined under microscope.
7
Hemolymph Phagocytic Activity Evaluation
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Hemolymph was collected after B. alexandrina snails were treated with sublethal concentration of NaOCl according to (Nduku and Harrison, 1980 ). Where, a capillary tube was used to draw hemolymph from a small opening above the snail's heart.
Hemocytes monolayers:
A glass slide with 10 μl of hemolymph was placed on it, and it was left at room temperature for 15 min to dry. Absolute methanol was used to fix hemocytes for 5 min, and 10% Giemsa stain (Aldrich) was used to stain them for 20 min., then examined under light microscope (Abdul-Salam and Michelson, 1980 (link)).
Phagocytic activity
Hemocyte suspension (100 μl) was collected from each exposed and control groups, then It was applied on glass slides and left to sit for one hour at 37 °C in a humid environment to test for cell adhesion. Hemocytes were challenged with activated charcoal particles and incubated for 1 h at 37 °C in order to measure their phagocytic efficacy. The cells were then stained with Giemsa after incubation, air dried, fixed with methanol, and viewed under a light microscope. Both in control and exposed snails, various stages of hemocyte phagocytosis of charcoal particles were identified. To estimate phagocytic index, the following formula was applied according to (Guria, 2018 ).
8
6-TG Breast Cancer Cell Colony Assay
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MCF-7 breast cancer cells (200) were cultured in 6-well plates at 37°C and 5% CO2 for 24 h. Next, the cells were washed in PBS and treated with 6-TG in a 37°C cell incubator. After 8 days, the cells were stained with 1× Giemsa stain (Sigma G4507) after being washed 3 times with PBS and fixed with methanol for 5 min. Finally, the colony formation numbers were counted under an inverted microscope.
9
Giemsa Staining Cell Colony Assay
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Cells were seeded in 6-well plates at a density of 2000 cells/well. After two-week culture, cells were fixed in 4% paraformaldehyde followed by 1 × Giemsa stain (Sigma Aldrich, St Louis, MO, USA). Excess Giemsa was removed by running water. Images were captured and cell colonies were counted using the Gel Doc XR system (Bio-Rad Laboratories, Hercules, CA, USA).
10
Cell Colony Quantification Protocol
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Cells were seeded in 12-well plates at a concentration of 5 × 103 cells/well. After a 10-day culture, cells were fixed in 4% paraformaldehyde followed by 1 × Giemsa stain (Sigma Aldrich, Saint Louis, MO, USA). Excess Giemsa was removed by rinsing the plate with running water. Images were then captured and cell colonies were counted using the Gel Doc XR system (Bio-Rad Laboratories, Hercules, CA, USA).
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