RFe iPSCs were treated with 100 ng/ml Colcemid solution (Life Technologies, 15210040) for 16 hours, then treated with 0.05% trypsin-EDTA for 15 minutes and filtered through a 40 μm cell strainer to remove clumps. RFe cells were collected by centrifugation, resuspended in 1 ml 0.075 M potassium chloride (Sigma-Aldrich, P9327) and incubated for 20 minutes at room temperature. 0.5 ml fixative [1 part glacial acetic (Fisher Scientific, A38-212) mixed with 3 parts methanol (Sigma-Aldrich, A412-4)] were added, RFe cells were collected as before, resuspended in 4 ml fixative, and incubated for 20 minutes at room temperature. The fixation step was repeated, the RFe cells collected as before and all but about 200 μl of the fixative was removed. The cells were resuspended in the remaining fixative and dropped onto slides that were precooled at −20°C. The slides were airdried and the RFe cells stained for 10 minutes with Giemsa Staining solution consisting of 1 part Giemsa solution (Life Technologies, 10092013), and 3 parts Gurr buffer (Invitrogen, 10582013). The slides were washed with water, dried, and mounted in Cytoseal 60 (Thermo Scientific, 23-244257). High-resolution pictures of chromosome spreads were acquired with an AxioObserver microscope (Zeiss) using the 100x oil objective.
Giemsa solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Giemsa solution is a laboratory reagent used in microscopy for the staining of blood smears and other cellular samples. It is a mixture of methylene blue, eosin, and azure dyes that selectively stain different cellular components, enabling the differentiation and identification of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cytoseal 60, by Thermo Fisher Scientific (2 mentions)
Colcemid solution, by Thermo Fisher Scientific (2 mentions)
P9327, by Merck Group (2 mentions)
A38 212, by Merck Group (2 mentions)
A412 4, by Merck Group (2 mentions)
Gurr buffer, by Thermo Fisher Scientific (2 mentions)
Axio observer microscope, by Zeiss (2 mentions)
Colchicine, by Merck Group (2 mentions)
Tcs sp5 confocal microscope system, by Leica (1 mentions)
Quantity one analysis software, by Bio-Rad (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse antibody, by Santa Cruz Biotechnology (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Gurr s buffer, by Thermo Fisher Scientific (1 mentions)
Karyomax colcemid solution in pbs, by Thermo Fisher Scientific (1 mentions)
Gsl120, by Leica (1 mentions)
Karyomax colcemid solution in, by Thermo Fisher Scientific (1 mentions)
11 protocols using giemsa solution
1
Karyotyping Induced Pluripotent Stem Cells
2
Karyotyping Induced Pluripotent Stem Cells
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RFe iPSCs were treated with 100 ng/ml Colcemid solution (Life Technologies, 15210040) for 16 hours, then treated with 0.05% trypsin-EDTA for 15 minutes and filtered through a 40 μm cell strainer to remove clumps. RFe cells were collected by centrifugation, resuspended in 1 ml 0.075 M potassium chloride (Sigma-Aldrich, P9327) and incubated for 20 minutes at room temperature. 0.5 ml fixative [1 part glacial acetic (Fisher Scientific, A38-212) mixed with 3 parts methanol (Sigma-Aldrich, A412-4)] were added, RFe cells were collected as before, resuspended in 4 ml fixative, and incubated for 20 minutes at room temperature. The fixation step was repeated, the RFe cells collected as before and all but about 200 μl of the fixative was removed. The cells were resuspended in the remaining fixative and dropped onto slides that were precooled at −20°C. The slides were airdried and the RFe cells stained for 10 minutes with Giemsa Staining solution consisting of 1 part Giemsa solution (Life Technologies, 10092013), and 3 parts Gurr buffer (Invitrogen, 10582013). The slides were washed with water, dried, and mounted in Cytoseal 60 (Thermo Scientific, 23-244257). High-resolution pictures of chromosome spreads were acquired with an AxioObserver microscope (Zeiss) using the 100x oil objective.
3
Undifferentiated Tree Shrew SPG Karyotyping
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Undifferentiated tree shrew SPGs were incubated with 0.25 μg/mL Colchicine (Sigma-Aldrich, C3915-500MG, USA) for 4 h at 37 °C in a 5% CO2 atmosphere in the incubator. Cells were collected and cell pellets were gently resuspended with 0.4% KCL (Sigma, Lot# SLBH5524V, USA)/0.4% sodium citrate (Aladdin, S189183-100g, China) (ratio 1:1) and incubated for 15 min at room temperature, followed by fixation with methanol (DM, 104119, China)/glacial acetic acid (Aladdin, Lot# I1712010, China) (ratio 3:1). Fixed cells were dropped onto microscope slides (CITOTEST, 188105W, China), air dried, and baked at 65 °C overnight. The slides were then stained with Giemsa solution (Life Technologies, 10092013, USA) (PBS/Giemsa=250:1) for 10 min and examined using the Leica TCS SP5 confocal microscope system (Leica Microsystems, Germany). At least 40 metaphase spreads were examined in three independent replicates.
4
3D Cell Culture Proliferation Assay
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The assay was conducted on 6-well plates. Each well contained a bottom layer of 1%
agarose, a middle layer of 0.5% agarose that included 7.5×103 stably
transfected SH-SY5Y cells, and a top layer of media. The media in the top layer was
changed every sixth day. After 14 days, cells were stained with Giemsa solution
(Gibco BRL) and counted using the Quantity One¯ analysis software (Bio-Rad
Inc.).
agarose, a middle layer of 0.5% agarose that included 7.5×103 stably
transfected SH-SY5Y cells, and a top layer of media. The media in the top layer was
changed every sixth day. After 14 days, cells were stained with Giemsa solution
(Gibco BRL) and counted using the Quantity One¯ analysis software (Bio-Rad
Inc.).
5
Quantification of Bacterial Adherence to HeLa Cells
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The HeLa epithelial cell line was maintained in DMEM-HEPES with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C under 5% CO2. HeLa cells were seeded into 6-well plates (108 cells/well) and grown for 24 hours. Subsequently cells were washed, and 3 ml of fresh complete medium without antibiotics was added to the wells. Cells were infected with 109 bacteria per well for 5 hours. After 3 washes with PBS, cells were fixed using methanol for 15 minutes and stained with 10% Giemsa solution (Gibco) for 20 minutes. The number of adherent bacteria per 25 cells was determined by direct count under a light microscope (Magnification 640 ×). The TTSS ΔescN strain [56] (link) was used as negative control.
6
Evaluating EGFR-Akt-P38 Signaling Pathway
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High glucose medium DMEM, 0.25% trypsin, PBS, penicillin-streptomycin solution, and Giemsa solution were obtained from Gibco Company. Fetal calf serum (FCS) was purchased from Sijiqing Company, Hangzhou. DMSO and Annexin V-FITC /PI cell apoptosis kit were purchased from Kaiji Company, Nanjing. Mouse monoclonal antibody against p-EGFR, p-Akt, and p-P38, mouse monoclonal antibody against β-actin, and horseradish peroxidase (HRP)-conjugated anti-mouse antibody were purchased from Santa Cruz Company. C225 (2mg/ml) was provided by the German Merck Company, Flow cytometry (FACS Calibur, Beckman Coulter Company). Linear accelerate (ELEKTA Company).
7
Chromosome Aberration Analysis of Hypoxic Cells
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Cells were synchronized into the G1 phase by mitotic shake off and 2 h of incubation. After 64Cu-ATSM was added, cells were incubated for 3 h under hypoxic conditions, then 0.1 μg/ml Colcemid (KaryoMAX® Colcemid™ Solution in PBS, Gibco) was added and incubated for 12–16 h under normoxic conditions. Since we observed a wide variety in a degree of chromosome aberrations after 64Cu-ATSM exposure, we assumed faster entry of less damaged cells and slower entry of heavily damaged cells to mitosis. This prolonged Colcemid treatment was aimed to collect a maximum population in metaphase cells. Harvested cells were treated with hypotonic solution (75 mM KCl) for 20 min at 37°C and fixed with methanol:acetic acid (3:1) solution three times before being dropped onto slides. Samples were stained with 5% (v/v) Giemsa solution in Gurr's buffer (Gibco). At least 50 metaphase cells were scored in at least two separate experiments. Chromosomal aberrations were quantified and classified as various chromosome and chromatid type aberrations.
8
Cytogenetic Analysis of MM9H-1 Cells
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The karyotype of MM9H‐1 cells was analyzed by GTG‐banding techniques (G‐bands by trypsin using Giemsa) using standard protocols. In brief, when cells had grown to 70% confluence, the cells were fed with fresh medium containing 0.2 μg/mL colchicine (Biological Industries, Beit HaEmek, Israel), and the culture was continued for an additional 4 h. Then, cells were harvested and treated with hypotonic solution (0.075 mol/L KCl) for 20 min at 37°C. The cells were fixed in freshly prepared methanol/glacial acetic acid (3:1) solution before they were evenly spread on a pre‐cooled slide and stained with Giemsa solution (Gibco). The slides were scanned with an automated slide scanner (GSL120, Leica, Nussloch, Germany) and analyzed with Applied Imaging Software CytoVision (Applied Imaging, Santa Clara, CA, USA).
9
Cytogenetic Analysis of Fibroblast Cell Lines
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After the cells reached 70% confluence, we added
KaryoMAX® Colcemid™ Solution in PBS-10 µg/ml (Gibco,
USA) to each flask to a final dilution of 25 µl/ml, which was
then incubated at 37°C for 45 minutes. We monitored the
changes in cell morphology with an inverted microscope
until the fibroblasts detached. For hypotonic treatment, we
slowly and carefully added 13 ml of 0.056% KCl (Merck,
Germany) with distilled water, followed by incubation at
37°C for 11 minutes, and fixed with methanol: acetic acid
(3:1) solution. In order to obtain G-bands, we aged the slides
at 60°C overnight. We carried out the staining procedure using
Giemsa solution 1:10 (Gibco, USA). Whenever possible, we
analysed 15 metaphases. Before printing out each karyotype
and counting each chromosome by writing a number on each
sister chromatid pair, we observed the slides under a light
microscope at ×10 and ×100 magnifications.
KaryoMAX® Colcemid™ Solution in PBS-10 µg/ml (Gibco,
USA) to each flask to a final dilution of 25 µl/ml, which was
then incubated at 37°C for 45 minutes. We monitored the
changes in cell morphology with an inverted microscope
until the fibroblasts detached. For hypotonic treatment, we
slowly and carefully added 13 ml of 0.056% KCl (Merck,
Germany) with distilled water, followed by incubation at
37°C for 11 minutes, and fixed with methanol: acetic acid
(3:1) solution. In order to obtain G-bands, we aged the slides
at 60°C overnight. We carried out the staining procedure using
Giemsa solution 1:10 (Gibco, USA). Whenever possible, we
analysed 15 metaphases. Before printing out each karyotype
and counting each chromosome by writing a number on each
sister chromatid pair, we observed the slides under a light
microscope at ×10 and ×100 magnifications.
10
Giemsa Staining for Cancer Cell Identification
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To confirm the presence of the cancer cells in the doped blood samples a Giemsa stain was used. Giemsa stain is a differential stain containing a mixture of azure blue, methylene blue, and eosin dye. Pathology laboratories commonly use Giemsa staining for blood work such as leukaemia and malaria diagnosis. A staining solution was prepared by diluting a stock Giemsa solution (Atom Scientific) (methanol <25%, glycerol <25%, ethylene glycol <25% and Giemsa powder) at a ratio of 1:40 with a Gurr buffer (Giemsa solution: Gurr buffer) (Thermo Fisher Scientific). The Gurr buffer was produced by adding the Gurr buffer tablet to 100 mL of distilled water as per the manufacturer instructions to produce a pH 6.8 phosphate buffer. The staining solution was dropped onto the sample area of the coverslips and incubated at room temperature for 45 minutes. After this time period, the staining solution was poured off the coverslips and any excess stain was washed off with the Gurr buffer. The samples were air dried to remove excess moisture from the washing. The areas of the samples that were measured with FTIR microspectroscopy were imaged under a microscope to confirm the identity of the cancer cells.
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