SaOS-2 and SaOS-LM2 cell lines (106) were cultured as described above and plated in 60 mm dishes (Fisher Scientific, Cat.# 08772B) for 24 h before any drug intervention to ensure adhesion of cells. Plated cells were then treated with enzalutamide, at IC50 concentrations (SaOS-2: 109.7 µM and SaOS-LM2: 98.46 µM) for 48 and 72 h. Each group included three technical triplicates for analyses. At the end of the treatment periods, cells were harvested with Tryple Express (Fisher Scientific, Cat.# 12604021). The ALDEFLUOR™ kit (Stemcell Technologies, Cambridge, MA, USA, Cat.# 01700) was used to analyze the cells with ALDH enzymatic activity according to the manufacturer’s protocol as described previously [38 (link)]. Briefly, cells were incubated in the ALDEFLUOR™ assay buffer containing the ALDH substrate BAAA at 37 °C for 45 min. 7- Aminoactinomycin D (7-AAD) dye (Thermo Fisher Scientific, Cat.# A1310) was added to stain dead cells. Stained cells were analyzed using BD LSR Fortessa (BD Biosciences, San Jose, CA, USA) with Flowjo software (Version 10.5.2+, Flowjo LLC). Three separate ALDEFLUOR™ assays were performed.
60 mm dishes
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
The 60 mm dishes are a versatile laboratory equipment item designed for a variety of cell culture and experimental applications. These dishes provide a standard-sized circular surface area for culturing cells, performing assays, or conducting other experimental procedures. The 60 mm size offers a convenient and practical solution for many common laboratory needs.
Automatically generated - may contain errors
Lab products found in correlation
Tryple express, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Irdye 800cw conjugated goat anti mouse, by LI COR (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Nocodazole, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
Stealth sirna, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Stealth rnai, by Thermo Fisher Scientific (1 mentions)
Axiovert 25, by Zeiss (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
8 protocols using 60 mm dishes
1
Evaluating ALDH Activity in Osteosarcoma Cells
2
Immunoblotting of S. cerevisiae and HeLa Cells
3
Apoptosis Assay in PC12 Cells
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For PC12-wt and PC12-AQP3 1×106 cells were plated on 60mm dishes (Nunc) during 48 h. After this time cells were treated with 7.5 μM of nocodazole (Sigma) for 24 h. Following, cells were harvested and washed with FACS, and stained with the commercial kit Annexin-V-FITC apoptosis detection kit (Inmunostep) to analyze alive, early and late apoptotic and necrotic cells by flow cytometry using a BD LSR Fortessa flow cytometer (BD Biosciences).
4
SN56 Cell Protein Analysis
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SN56 cells (1.5 x 106) were plated on 60 mm dishes (Nunclon) and transfected with the appropriate DNA constructs/siRNA transcripts. Following 3 days of incubation, cells were washed in cold PBS and lysed with NP40 lysis buffer (20 mM Tris pH 8.0, 137 mM NaCl, 10 % glycerol, 1 % IGEPAL/NP40) for 5 min at 4 °C. Cells were then scraped and centrifuged at 14,000 rpm for 15 min at 4 °C to remove insoluble material. Total cell lysates were separated by SDS-PAGE and transferred onto PVDF (polyvinylidene fluoride) membrane.
PVDF membrane was then probed with Arf1 (1:1000), Arf6 (1:1000) or α-tubulin (1:10,000) antibodies (Sigma), developed using ECL and exposed to film. Quantification of western blot exposures was accomplished using ImageJ.
PVDF membrane was then probed with Arf1 (1:1000), Arf6 (1:1000) or α-tubulin (1:10,000) antibodies (Sigma), developed using ECL and exposed to film. Quantification of western blot exposures was accomplished using ImageJ.
5
Silencing NS Gene in HOC-313 Cells
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HOC-313 cells were used in this experiment. Twenty-four hours before small interfering RNA (siRNA) transfection, the cells were diluted in fresh medium without antibiotics and were transferred to 60-mm dishes (Nunc, Waltham, MA, USA). The cells were grown and transfected with NS-specific siRNA and Stealth RNAi Universal negative control (40 nM , Stealth siRNA, Invitrogen, Carlsbad, CA, USA) using Lipofectamine RNAi MAX (Invitrogen), as described in the manufacturer's instructions. The sequences for siRNA were as follows: for NS, sense strand 5′-UCGCAUUGACCAUUCAACUGGUGG-3′, antisense strand 5′-CCACCAGUUUGAAUGGUCAAU-3′(named NS1), and sense strand 5′-UCGCAUUGACCAUUCAACUGGUGG-3′, antisense strand 5′-CCACCAGUUUGAAUGGUCAAU-3′ (named NS2). The cells were harvested 48 h after transfection for a western blot analysis.
6
TGF-β1 Induced Morphological Changes
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A549 (2.4 × 105), MRC-5 (3 × 105), and LL-29 (2.5 × 105) cells were plated on 60 mm dishes (Nunc, Roskilde, Denmark), when the cells reached 80% of confluence we added fresh medium 1% FBS for 24 h and then we added 10 ng/ml of TGF-β1 (R&D System, Minneapolis, MN, USA) for 4, 16, 24, 48, or 72 h. We used untreated cells as control.
Morphological changes in A549, MRC-5 and LL-29 cells were examined by inverted fluorescence microscopy (Zeiss Axiovert 25, Carl Zeiss Co. Oberkochen, Germany) and photographed using a CCD Canon camera.
Morphological changes in A549, MRC-5 and LL-29 cells were examined by inverted fluorescence microscopy (Zeiss Axiovert 25, Carl Zeiss Co. Oberkochen, Germany) and photographed using a CCD Canon camera.
7
Isolation of Microglia and Astrocytes
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To obtain microglia and astrocytes in vitro, we prepared primary mother cultures of the cerebral hemispheres of newborn rat. The primary cultures were made essentially according to a method described previously,27 (link)
and both microglia and astrocytes were prepared from the cultures.
Briefly, microglia were floated by gentle shaking of primary mother cultures that had been maintained for 10–20 days, and seeded on 60-mm dishes (Nunclon, Sigma-Aldrich) at a density of 1.5 × 106 (for immunoblotting). The purity was over 99.9% based on the assessment of Iba1 staining.
Astrocytes were prepared from a primary mother culture maintained for 3 weeks, essentially as described.28 (link)
The astrocytes were subcultured onto 60-mm dishes (Nunclon) at a density of 1.5 × 106 cells/dish (for immunoblotting). The cell purity was estimated as 98% based on staining with anti-GFAP antibody.
and both microglia and astrocytes were prepared from the cultures.
Briefly, microglia were floated by gentle shaking of primary mother cultures that had been maintained for 10–20 days, and seeded on 60-mm dishes (Nunclon, Sigma-Aldrich) at a density of 1.5 × 106 (for immunoblotting). The purity was over 99.9% based on the assessment of Iba1 staining.
Astrocytes were prepared from a primary mother culture maintained for 3 weeks, essentially as described.28 (link)
The astrocytes were subcultured onto 60-mm dishes (Nunclon) at a density of 1.5 × 106 cells/dish (for immunoblotting). The cell purity was estimated as 98% based on staining with anti-GFAP antibody.
8
Clonogenic Assay for MNBE Impact
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To determine the effect of MNBE on colony formation ability of HeLa, SiHa, and ME-180 cells, clonogenic assay was performed. Viable cells were plated in the range of 200–1000 cells depending on the cell type in 60 mm dishes (Nunc, Denmark) and allowed to attach for 24 h. After 24 h, the cells were treated with MNBE at their respective IC25 and IC50 dose or vehicle (0.1% DMSO) and incubated further for 24 h in a humidified CO2 incubator. After 24 h, media was replaced with fresh medium and incubated for another 10–14 days to form colonies. Cells were then fixed with 4% paraformaldehyde and stained with 0.4% crystal violet. Colonies with at least 50 cells were counted manually, and the survival fraction was calculated by dividing the number of colonies that arise after treatment by the number of cells seeded and plating efficiency (PE: number of colonies formed by untreated cells/number of cells seeded) [22 (link)]. All treatments were performed in triplicate, and the experiments were repeated three times.
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