bleomycin and cisplatin concentrations were chosen to match those used in a previous study [8 (link)]. A 10 mg vial of bleomycin (Hospira, Lake Forest, IL, USA ) was reconstituted in sterile 0.9% w/v sodium chloride and stocks frozen at −20 °C. Revived PBMCs were resuspended in serum-free media RPMI-1640 (Gibco, USA) in six-well Nunclon™ Delta plates (Thermo Scientific, USA) containing 2.5 × 106 cells per well with bleomycin at concentrations of 10, 20, 30, or 40 μM or vehicle (0.9% w/v sodium chloride). Plates were incubated for 30 min at 37 °C in 5% CO2, after which cells were washed in PBS and prepared for DNA extraction. A 1 mg/mL stock solution of cisplatin (Novartis, Auckland, New Zealand) was diluted to the required concentration in complete media RPMI-1640 (Gibco, USA). Revived PBMCs were resuspended in complete media RPMI-1640 (Gibco, USA) in six-well Nunclon™ Delta plates (Thermo Scientific, USA) containing 2.5 × 106 cells per well with cisplatin at concentrations of 50 or 100 μM or media control. Plates were incubated for 24 h at 37 °C in 5% CO2, after which cells were washed in PBS and prepared for DNA extraction.
Nunclon delta plates
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunclon Delta plates are a type of cell culture plates designed for adherent cell growth. They feature a treated surface that promotes cell attachment and proliferation. The plates are available in a range of sizes and well configurations to accommodate different experimental needs.
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Lab products found in correlation
Fluostar omega, by BMG Labtech (1 mentions)
Bleomycin, by Thermo Fisher Scientific (1 mentions)
Bleomycin, by Pfizer (1 mentions)
Cisplatin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
6 well ultra low attachment culture plates, by Corning (1 mentions)
A2780, by Corning (1 mentions)
B27 additive, by Thermo Fisher Scientific (1 mentions)
A2780, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
5 protocols using nunclon delta plates
1
Cytotoxicity Assay with Bleomycin and Cisplatin
2
Quantifying GFP Fluorescence in ISF
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For GFP quantification, 100 μL ISF buffer duplicates were added to black plastic 96 well Nunclon Delta plates (Thermo #137101). The samples were measured for fluorescence intensity with a FLUOstar Omega plate reader using Omega control and data analysis software (BMG Labtech). The excitation wavelength was set at 480 nm, and the emission wavelength set at 520 nm. Data was transferred to Microsoft Excel for data analysis.
3
PBMC Stimulation and Cytokine Quantification
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PBMCs were first rested for 30 minutes in RPMI 1640 + 10% heat-inactivated FCS + Penicillin-Streptomycin. Then, 4.10e5 PBMCs were plated in 96-well round bottom Nunclon Delta plates (Thermo Fisher Scientific) and stimulated with LPS (10 ng/mL), Pam3Cys (10 µg/mL) or poly I:C HMW (5 µg/mL) for 24 hours or IFN-β1a (100 U/mL) for 0.5, 2 or 6 hours (final volume 200 µl) in a humidified incubator at 37°C/5% CO2. Harvested supernatants were kept on ice at all times and immediately stored at -20°C until quantification of cytokine levels, or stored at -80°C until lactate and glucose measurements. Cells were lysed in RLT buffer and stored at -20°C until RNA isolation.
4
Ovarian Cancer Cell Culture Protocol
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The human epithelial ovarian cancer cell line, A2780, was purchased from Sigma. The human ovarian adenocarcinoma cell line, 3AO, was obtained from Women's Hospital, School of Medicine, Zhejiang University, which was tested and authenticated. 3AO and A2780 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (BI, Kibbutz Beit-Haemek, Israel), supplemented with 10% fetal bovine serum (FBS) (Invitrogen, New York, NY, USA) at 37°C and 5% CO2. The adherent cells were cultured in serum-free medium (SFM) composed of 10uL/mL B27 additive (Life Technologies, Carlsbad, CA, USA), 10 ng/mL, 1 mg/mL insulin (Sigma-Aldrich, Burlington, MA, USA), basic fibroblast growth factor, 20 ng/mL epidermal growth factor (Pepro-Tech, Rocky Hill, CT, USA), Dulbecco's modified Eagle's medium (DMEM/F12) (BI), to form spheroids after plating 5×104 cells per well in ultra-low attachment 6-well culture plates (Corning, New York, NY, USA). The culture medium will be renewed every two or three days. After plating 400 or 600 cells per well in ultra-low attachment 96-well culture plates (Corning), A2780 or 3AO cells were cultured in SFM at 37°C in 5% CO2 for 7 days. The culture medium will be renewed every two or three days. The spheroids were cultured in RPMI-1640 medium with 10% FBS after plating in 6 Nunclon Delta plates (Thermo Scientific, Suzhou, China) to re-differentiate.
5
UVC Exposure Effects on Cell Lines
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UVC irradiation was carried out on THP1, A549, and fresh or cryopreserved PBMC cells. Cell concentrations were 2.5–5 × 105/mL for A549 cells, 5 × 105/mL for THP1, and 1 × 106/mL for PBMCs, aliquoted into identical 2 mL media volumes in six-well Nunclon™ Delta plates (Thermo Scientific, USA). UVC doses of either 20 or 100 millijoules per cm2 (mJ/cm2) were given to each plate using the Bio-link BXL-254 cross-linker (Gibco, USA). Cells were harvested immediately after UVC exposure, and were prepared for DNA extraction, except for aliquots of fresh PBMCs that were used within the alkaline comet assay.
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