hscNPCs and hscAS at passage 5 were treated with 2 μg/ml colcemid solution (10092013, Invitrogen) for 4 h at 37°C following metaphase arrest. Then cells were dissociated and resuspended in a hypotonic solution of 0.00375 M KCl for 10 min at RT. Centrifugation and fixation were repeat 3 times. Dried samples were stained with DAPI mounting medium (ZLI‐9557, ZSGB‐BIO) and harvest of chromosome clusters was confirmed under a fluorescence microscope. The definition of clones and the description of karyotypes were in accordance with International System for Human Cytogenetic Nomenclature (ISCN, 2006) (n = 3 samples).
Colcemid solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Colcemid solution is a laboratory reagent used to arrest cell division at metaphase. It functions by inhibiting the polymerization of microtubules, which are essential for cell division. This solution is commonly used in cytogenetic analyses and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Shandon cytospin 4, by Thermo Fisher Scientific (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Phytohemagglutinin (pha), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Metafer imaging platform, by MetaSystems (2 mentions)
Giemsa solution, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Metafer4, by MetaSystems (2 mentions)
Cytoseal 60, 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)
Giemsa solution, by Thermo Fisher Scientific (2 mentions)
Axio observer microscope, by Zeiss (2 mentions)
Ikaros software, by MetaSystems (1 mentions)
Cd11b fitc, by Merck Group (1 mentions)
Cd34 fitc, by Merck Group (1 mentions)
Cd44 fitc, by Merck Group (1 mentions)
Cd45 pe, by Merck Group (1 mentions)
23 protocols using colcemid solution
1
Chromosome Analysis of hscNPCs and hscAS
2
Karyotyping of OVPA8 Cells via G-banding
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G-banding was performed according to classical protocol for adherent cells [41 (link)]. Briefly, OVPA8 cell cultures in their logarithmic growth phase were exposed for 45 min. to Colcemid solution (Invitrogen) at final concentration of 0.1 µg/mL. Afterwards, the cells were treated with pre-warmed hypotonic lysis solution (0.075 M KCl) at 37 °C for 10 min and fixed with Carnoy’s solution (3:1 methanol:glacial acetic acid) at room temperature. Cell suspension was dropped onto glass slides, air-dried and treated by pre-warmed 0.25% trypsin solution at 37 °C for 3–10 s. Staining was done using 4% Giemsa solution in Sorensen buffer for 10 min. Slides were rinsed in water and air-dried at room temperature. Cytogenic analysis was done using Ikaros software (MetaSystems, Heidelberg, Germany). Seven metaphase spreads were analyzed and chromosomes were classified according to the International System for Human Cytogenetic Nomenclature (ISCN).
3
Chromosome Spread Analysis of iPSCs
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The division of iPSCs was blocked with 50 µg/mL of colcemid solution (Invitrogen, USA). Cells were washed with PBS and harvested with trypsin at room temperature for 2 min. Then cells were fixed in methanol/glacial acetic acid (3:1) for three times and dropped onto slides for chromosome spreads. At last, the slides were baked at 55 °C for overnight. Standard G-banded karyotypes were obtained using Giemsa solution staining (Giemsa, Japan).
4
Isolation and Characterization of Stem Cells
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We used the following materials in this clinical
trial study: collagenase A type I (Sigma, USA), fetal
bovine serum (FBS, Gibco, USA), MEM Alpha
1x (Gibco, USA), L-glutamine (Gibco, USA),
antibiotic-antimycotic solution (Gibco, USA),
trypsin-EDTA (Gibco, USA), CD90-fluorescein
isothiocyanate (FITC), CD73-phycoerythrin (PE),
CD105-PE, CD34-FITC, CD45-PE, CD11b-FITC,
CD44-FITC (Sigma-Aldrich, USA), colcemid
solution (Invitrogen, USA), and dimethylsulfoxide
(DMSO, Gibco, USA). One-way ANOVA test was
used in this study which was not significant (P>0.05).
trial study: collagenase A type I (Sigma, USA), fetal
bovine serum (FBS, Gibco, USA), MEM Alpha
1x (Gibco, USA), L-glutamine (Gibco, USA),
antibiotic-antimycotic solution (Gibco, USA),
trypsin-EDTA (Gibco, USA), CD90-fluorescein
isothiocyanate (FITC), CD73-phycoerythrin (PE),
CD105-PE, CD34-FITC, CD45-PE, CD11b-FITC,
CD44-FITC (Sigma-Aldrich, USA), colcemid
solution (Invitrogen, USA), and dimethylsulfoxide
(DMSO, Gibco, USA). One-way ANOVA test was
used in this study which was not significant (P>0.05).
5
Chromosomal Analysis of Synchronized iPSCs
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The iPSCs, grown in Matrigel-coated dishes, were synchronized using 0.1 µg/ml of Colcemid solution (Invitrogen). After fixation with fresh methyl alcohol/glacial acetic acid (3:1; Sigma-Aldrich), a small quantity of the cells was spread onto 10 slides. Chromosomes were examined, counted by phase-contrast microscopy, and photographed (200×).
6
Chromosome Counting Using Metafer Imaging
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Cells grown to ∼ 80% confluency were treated with 100 ng/ml Colcemid solution (Gibco) for 3 h, collected by trypsinization, and centrifuged at 1,000 rpm for 10 min (Giam et al, 2020 (link)). Cell pellets were resuspended in 75 mM potassium chloride solution and incubated for 15 min in a 37°C waterbath. Next, a 1/10 volume of 3:1 methanol/acetic acid was added to the cells prior to centrifugation at 1,000 rpm for 15 min. Cells were then fixed by resuspension in 3:1 methanol/acetic acid solution, incubated for 30 min at room temperature, centrifuged at 1,200 rpm for 5 min, and finally washed once more with fixative. Cells were resuspended in a small volume of fixative, dropped onto clean glass slides, and allowed to air‐dry. For chromosome counting, metaphase spreads were stained with Giemsa stain (Gibco) and acquired using the fully automated Metafer imaging platform (MetaSystems). Chromosome numbers were scored manually using ImageJ.
7
Chromosome Preparation of CFSC-2G Cell Line
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The preparation of chromosomes of CFSC-2G was essentially conducted as published before [6 (link)]. In brief, semi-confluent CFSC-2G cultures were exposed to colcemid solution (Gibco, ThermoFisher Scientific, Dreieich, Germany), detached, and harvested by centrifugation. Thereafter, the cells were treated with 0.56% (w/v) hypotonic potassium chloride solution and fixed with a mixture of methanol and acetic acid (3:1). Air-dried chromosome spreads were prepared, and slides were treated with 0.025% (w/v) trypsin solution followed by staining with Giemsa solution to visualize G-banding pattern. In addition, the heterochromatin in metaphase chromosomes was stained following an established procedure [11 (link)]. At least ten metaphases were analyzed from the GTG-stained (G-bands by trypsin using Giemsa) and CBG-stained (C-bands by Barium hydroxide using Giemsa) slides.
8
Mitotic Arrest and Chromosome Preparation
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To induce mitotic arrest, approximately 1 × 105 cells were treated with 0.5 μg/mL Colcemid Solution (Gibco/ThermoFisher) for 2 h in a 28°C heat block. Cells were then spun for 5 min at 600x g at RT to pellet and resuspended in hypotonic solution (500 mL of 0.5% sodium citrate). Cells were incubated in hypotonic solution for 8 min at RT. 100 μL of the cell suspension was then placed in a cytofunnel and spun at 1200 rpm for 5 min with high acceleration using a cytocentrifuge (Shandon Cytospin 4; ThermoFisher). For FISH, slides were then fixed in cold 3:1 methanol: acetic acid for 10 min and washed 3X 5 min in PBS-T (PBS with 0.1% Triton X-100). FISH was performed as described above for meiotic squashes.
9
Whole Blood Cytogenetic Analysis Protocol
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Samples were cultured according to the methods recommended by the IAEA (1 ). Briefly, whole blood was cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine serum (Gibco), 1% antibiotic-antimycotic (Gibco), and 2% phytohemagglutinin (Gibco) at 37°C for 48 h in a humidified atmosphere of 5% CO2 in air. Colcemid solution (Gibco), at a concentration of 0.07 μg/ml, was added to the medium, 24 h before harvesting. Blood samples were treated with 0.075 M KCl for 25 min at 37°C, followed by fixation with cold methanol and acetic acid (3:1). Fixed cells were dropped onto slides, which were stored for 12 h at 60°C. The slides were stained with Giemsa solution (Sigma-Aldrich, St Louis, MO, USA). Metaphase images were captured using Metafer 4 (MetaSystems GmbH, Altlussheim, Germany).
10
Chromosomal Aberration Analysis Protocol
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Samples were cultured according to methods recommended by the IAEA [9 ] and ISO [10 ]. Briefly, whole blood was cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine serum (JRScientific Inc., CA, USA), 1% antibiotics (Gibco) and 2% phytohemagglutinin (Gibco) at 37°C for 48 h in a humidified atmosphere of 5% CO2 in air. Colcemid solution (Gibco) at a concentration of 0.07 μg/ml was added to the medium 24 h before harvesting. The fluorescence-plus-Giemsa (FPG) staining technique coupled with 15 μM BrdU (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in cultures and Hoechst 33 258 (Invitrogen) allows identification by the differential staining of second-division metaphase spreads. Blood samples were treated with 0.075 M KCl for 20 min at 37°C, followed by fixation with cold methanol and acetic acid (3:1). Fixed cells were spread onto slides, which were stored for 12 h at 60°C. The slides were stained with Giemsa solution (Sigma-Aldrich, St Louis, MO, USA). Metaphase images were captured using the Metafer 4 (Metasystems GmbH, Altlussheim, Germany) and analyzed by well-trained scorers.
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