Cells were seeded into 60 mm petri dishes (Primaria, Corning, US) and after a monolayer was formed, cell medium was replaced by SMCM supplemented with 40 μM or 120 μM Zn2+ solutions. After incubation for 24 h, total RNA was isolated by RNeasy Mini Kit (Qiagen, US). The concentration and purity of total RNA were determined by spectrophotometer (Nanodrop 2000, US) at 230 nm, 260 nm and 280 nm. The range of A260/A280 was 2.00–2.05 and the range of A260/A230 was 2.20–2.29. Then 500 ng total RNA was inversely transcripted into cDNA by RT2 First Strand Kit (Qiagen, US), according to the manufacturer’s protocol. RT-PCR was performed in a CFX96 Touch RT-PCR Detection System (Bio-Rad, US) using microarray. cDNA was mixed with RT2 SYBR Green Master Mix (Qiagen, US) and RNase-free water. In the 96-well PCR array plate, 25 μl of the mixture was added to each well. After 10 min incubation at 95 °C, cDNA was amplified according to the following parameters: denaturing for 15 s at 95 °C, then annealing for 1 min at 60 °C. 40 cycles were performed. After the amplification, Ct values were collected and analyzed by Bio-Rad CFX Manager 3.1 (Bio-Rad, US). The cutoff Ct value was 35. The ΔΔCt method was used to calculate the fold change.
60 mm petri dishes
Manufactured by Corning
Sourced in United States
The 60 mm Petri dishes are circular, shallow, and transparent containers used for cell culture, microbiology, and other laboratory applications. They are made of high-quality glass or polystyrene material and provide a controlled environment for the growth and observation of cells, microorganisms, or other samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Eclipse ts2, by Nikon (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Pet membrane, by BD (2 mentions)
Penicillin streptomycin, by Cytiva (2 mentions)
Cfx96 touch rt pcr detection system, by Bio-Rad (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Rt2 sybr green mastermix, by Qiagen (1 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
Fibroblast growth kit low serum, by American Type Culture Collection (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Las 4000 imager, by Fujifilm (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
14 protocols using 60 mm petri dishes
1
Zinc Modulates Gene Expression
2
Labeling Polymers for Cell Imaging
3
Immunophenotyping of Cell Cultures
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Cells were cultured in 60 mm Petri dishes (Corning, USA) at a density of 106 cells/dish overnight and then fixed with 80% methanol. Primary anti-CD34, anti-CD44, anti-CD45, and anti-CD146 rabbit monoclonal antibodies (Abcam, UK) were incubated with the cells at a concentration of 1 μg/106 cells for 30 min. The samples were next incubated using a goat anti-rabbit fluorescent secondary antibody (ABclonal, China) at a concentration of 1 μg/106 cells for 1 h and assessed using FACSCalibur flow cytometry (BD Biosciences, USA).
4
Wound Healing Assay for SK-MEL-5 Cells
5
Culturing Human Dermal Fibroblasts
6
Immunoblotting of Scraped Cells
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2×105 cells were plated in 60 mm petri dishes (Corning) 1 day prior to irradiation. Medium was removed and cells were washed once with PBS before harvesting by scraping directly in SDS loading buffer, transferred to screw cap microcentrifuge tubes, boiled for 5 minutes, then stored at −20°C until required. Samples were run on 10% SDS-PAGE gels (Biorad) before transferring to PVDF (Biorad). Membrane washes and antibody dilutions were performed using Tris-buffered saline with 0.1% (v/v) Tween20 (TBST). All primary antibody incubations were performed in TBST with 5% BSA, overnight at 4°C before washing, applying HRP-conjugated secondary antibody (NEB) for 1 h at room temperature and subsequently detecting HRP activity using Supersignal West Pico substrate (ThermoScientific, Cramlington, UK) with a LAS4000 imager (Fujifilm, Japan).
7
Isolation and Characterization of Mouse Cells
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Microsurgery scissors and forceps were purchased from Jiaxing Moore Trade Co. (Jiaxing, China), 0.2-µm syringe filters were purchased from Jet Bio-filtration (Guangzhou, China), and 1.5-ml Eppendorf tubes, 60-mm Petri dishes, 3-ml plastic pipettes and 75-cm2 flasks were purchased from Corning (Corning, NY, USA). Penicillin-streptomycin, Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Type IV collagenase, Tween-20, Triton X-100, phosphate-buffered saline (PBS) and bovine serum albumin (BSA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Purified rat anti-mouse CD106 (cat. no. 553330), purified rat immunoglobulin G2a (IgG2a), κ isotype control (cat. no. 553927), FITC goat anti-rat Ig secondary antibody (cat. no. 554016), phycoerythrin (PE) hamster anti-mouse CD54 (cat. no. 553253), PE hamster IgG1 κ isotype control (cat. no. 553972), biotin rat anti-mouse CD90.2 (cat. no. 553011), biotin rat IgG2b, κ isotype control (cat. no. 553987) and PE-streptavidin (cat. no. 554061) were obtained from BD Pharmingen (Franklin Lakes, NJ, USA), vimentin rabbit monoclonal antibody (Alexa Flour 488 Conjugate; cat. no. 9854) was obtained from Cell Signaling Technology (Beverly, MA, USA).
8
Isolation of Small Extracellular Vesicles from Breast Cancer Cell Lines
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The MCF-7 and MDA-MB-231 (ATCC) breast cancer cell lines were cultured in Dulbecco’s modified eagles medium (DMEM; Gibco, Thermo Fischer Scientific, Carlsbad CA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 100U/mL penicillin, and 100 μg/mL streptomycin, in a 5% CO2 humidified atmosphere at 37 °C. Cells were cultured in six 60 mm Petri dishes (Corning, CA) with FBS-originated-exosome-free media (as per protocol by Théry; FBS was ultra-centrifuged at 100,000×g for 2 h at 4 °C, then filtered with a 0.22 µm sterile filter). After 48 h incubation, the media containing sEVs were isolated. Total cell count was 2 × 107 and 24 mL of sEV-containing media was obtained. sEV isolation was performed as outlined in Fig. 1 . Successful sEV isolation was confirmed by electron microscopy, immune labeling (CD63, CD81 and CD9), and enrichment of sEV associated proteins determined using Mass Spectrometry (Supplemental Information).
9
Culturing Breast Cancer Cell Lines
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IBC cell lines SUM149, SUM190 and non-IBC cell lines MD-MB-231 and MCF-7 were purchased from ATCC (Manassas), Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, California) containing 10% fetal bovine serum (900-108, Gemini), penicillin G (MP Biomedicals, Irvine),and streptomycin (MP Biomedicals, Irvine) in an incubator at 37 °C with 5% CO2. Cells were seeded in 60-mm Petri dishes (Corning, NY, USA) at a density of 2.5×105 cell/dish and placed in an incubator at 37 °C with 5% CO2 for further culture. After that, the medium is changed every two days and passed on every 5 days. The Ethics Committee of the Third Xiangya Hospital of Central South University approved this study (No. 2017-S302). All experiments were conducted according to the experimental guidelines of the Third Xiangya Hospital, Central South University.
10
Wound Healing Assay for SK-MEL-5 Cells
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SK-MEL-5 cells were seeded in complete growth medium in 60 mm petri dishes (Corning) (0.25 ×106 cells/dish) in complete growth medium and allowed to attach in a 37 °C humidified 5% CO2 incubator for 1 week, resulting in a confluent monolayer of cells. The medium was removed, and cells were incubated overnight in the absence of FBS. The medium was removed, then cross-shaped scratches (wounds) were made on the cell monolayer with a sterile 200 μL pipette tip. The dishes were washed with DPBS, and cells were incubated in the presence of 1% EtOH (negative control) or 443 μg/mL (concentration equal to 5 x IC50 value, Fig. 1B ) of T. versicolor mycelium extract. The wound area was photographed with inverted phase contrast microscopy using a Nikon Eclipse Ts2 at time 0 and after 20, 24, and 48 h. Three biological replicates were performed, and seven measurements for each replicate were taken at each time point, followed by the calculation of mean ± SD. At each time point, the wound surface at that timepoint was divided by the wound surface measured at time 0 (defined as 100%) as a measure of wound closure.
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