Cells in the logarithmic growth phase were detached with trypsin and lightly dissociated into a cell suspension using a straw. The cells were inoculated into 6‐cm culture dishes with each culture dish containing 200 cells and incubated with complete culture medium containing 10 nmol/mL cisplatin in a 5% CO2 incubator at 37°C for 2‐3 weeks. During this period, the culture medium needed no replacement, and the culture was stopped when the clone was visible to the naked eye. With the culture medium discarded, the cells were washed two times with PBS, 5 mL methyl alcohol was added and allowed to stand at room temperature for 15 min. The fixation liquid was absorbed via a vacuum pump, and the Giemsa dye solution (SIGMA, USA) was added to the cells for 30 min. After the dye solution was discarded, the culture dish was air‐dried. The number of clones was calculated directly by the naked eye and the clone formation rate was calculated.
Giemsa dye solution
Manufactured by Merck Group
Sourced in United States
Giemsa dye solution is a laboratory reagent used for staining biological samples, particularly in the field of hematology. It is a combination of methylene blue, eosin, and azure dyes that selectively stain different cellular components, allowing for the visualization and differentiation of various blood cells and other cellular structures under a microscope.
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Lab products found in correlation
Propidium iodide, by Merck Group (1 mentions)
Matrigel, by Merck Group (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
Vitamin c, by Guangzhou Chemical (1 mentions)
Lysotracker, by Merck Group (1 mentions)
Annexin 5 fluorescein isothiocyanate annexin 5 fitc, by Merck Group (1 mentions)
4 6 diamino 2 phenylindole dapi, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by Merck Group (1 mentions)
Lc 20a hplc, by Shimadzu (1 mentions)
Transwell kit, by Corning (1 mentions)
Cck 8 kit, by Merck Group (1 mentions)
Dmem medium, by Corning (1 mentions)
Spd m20a detector, by Shimadzu (1 mentions)
5 protocols using giemsa dye solution
1
Clonogenic Assay for Cisplatin Sensitivity
2
miR-29b-3p Mimics Transfection Assay
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Using cells in the logarithmic growth phase, 100 cells/well were inoculated in 6-well plates at 37°C and cultured in a 5% CO2 incubator for 6 h. The cells were divided into three groups; one group was a blank control group without any treatment. The other two groups were transfected with mimics-NC (negative control) or miR-29b-3p mimics. The miR-29b-3p mimics and control were purchased from Shanghai Jima Pharmaceutical Technology Ltd. The plates were incubated at 37°C and 5% CO2 for 2–3 weeks. The culture was terminated when colonies were visible to the naked eye. The cells were washed twice with PBS and allowed to dry naturally. The cells were fixed with 4% methanol at room temperature for 15 min and were allowed to dry naturally after the methanol was discarded. The cells were stained with Giemsa dye solution (Sigma) for 10 min, washed slowly with running water, and air-dried.
3
Nano-Selenium Composite Cytotoxicity Assessment
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Sodium selenite (Na2SeO3), chitosan (CS), polyvinylpyrrolidone (PVP), lentinan (LET), and vitamin C (Vc) were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Thiazolyl blue tetrazolium bromide (MTT), propidium iodide (PI), 4’,6-diamino-2-phenylindole (DAPI), 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraacetyl carbocyanine iodide (JC-1), Lyso-tracker, Hoechst 33342, Annexin V-fluorescein isothiocyanate (Annexin V-FITC), vascular endothelial growth factor (VEGF), Matrigel, and Giemsa dye solution were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ultra-pure water, purified by Millipore’s Milli-Q water purification system, was used in all experiments.
4
Investigating RSF-1 in Breast Cancer Cells
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Michigan cancer foundation-7 (MCF-7) and SKBR-3 breast cancer cells purchased from (Cell Bank of the Chinese Academy of Sciences, Shanghai, China); Cell Counting Kit-8 (CCK-8), bromodeoxyuridine (BrdU) kits, and Giemsa dye solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); interferences RSF-1 small-interfering ribonucleic acid (siRNA), scrambled control siRNA and DharmaFECT 1 kits (Thermo Fisher Scientific, Inc., Waltham, MA, USA), RSF-1 human primary antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase (HRP)-labeled secondary antibodies (Abcam, Cambridge, MA, USA); Dulbecco's modified Eagles medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.); apoptosis kits, TRIzol kits (Invitrogen; Thermo Fisher Scientific, Inc.) and 4′,6-diamidino-2-phenylindole (DAPI) staining kits (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China). Rabbit monoclonal RSF-1 antibody (dilution 1:500; cat. no. ab109002), rabbit polyclonal GAPDH antibody (dilution 1:500; cat. no. ab37168) and secondary goat anti-rabbit (HRP) IgG antibody (dilution 1:2,000; cat. no. ab6721) were all purchased from Abcam. This study was approved by the Ethics Committee of China Medical University (Liaoning, China).
5
Cell Apoptosis Assay Protocol
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DMEM medium and Transwell kit were purchased from Corning Inc.; Fetal bovine serun (FBS) was purchased from Ausbian Inc., Australia; CCK-8 kit and Giemsa dye solution were purchased from Sigma Inc., USA; The Annexin V-FITC/PI double-stained cell apoptosis assay kit was purchased from eBioscience Inc., USA.
Ultra-clean workbench (Sujing Antai Company), CO2 incubator (Hitachi Company). LC-20A HPLC, including SPD-M20A detector, DGU-20A quaternary pump (Shimadzu company); KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)
Ultra-clean workbench (Sujing Antai Company), CO2 incubator (Hitachi Company). LC-20A HPLC, including SPD-M20A detector, DGU-20A quaternary pump (Shimadzu company); KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)
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