Different composites were inserted into 24-well plates (Sarstedt, Germany) and immersed in 1.1 mL culture medium for 24 h at 34 °C and 5% CO2 in CB240 incubator (Binder, Tuttlingen, Germany). After the incubation, the extract was collected. The day prior to the experiment, the hFOB 1.19 cells were seeded at the density of 5 × 104/cm2 on 24-well plates. On the next day, the conditioned media was replaced with the extracts, and the cells were incubated for 48 h at 34 °C and 5% CO2. After the incubation, cell viability was estimated by measuring the mitochondrial activity (WST-1 test) and cell cytotoxicity was measured by the leakage of lactate dehydrogenase (LDH cytotoxicity assay). All types of the composites were tested in a least 4 replicates. To obtain the extract, each tested sample was prepared from one disc.
Cb240 incubator
Manufactured by Binder
Sourced in Germany
The CB240 incubator is a laboratory equipment designed for controlled temperature and humidity environments. It maintains a stable temperature and humidity level within the internal chamber, providing a suitable environment for various applications such as cell culture, microbiology, and other temperature-sensitive experiments.
Automatically generated - may contain errors
Lab products found in correlation
24 well plate, by Sarstedt (2 mentions)
Hfob1.19 cells, by American Type Culture Collection (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Hfob 1, by LGC (1 mentions)
Flat bottom 96 well plate, by Sarstedt (1 mentions)
Wst 1 assay kit, by Abcam (1 mentions)
5 protocols using cb240 incubator
1
Cell Viability and Cytotoxicity Evaluation
2
Culturing Immortalized Fetal Osteoblasts
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Immortalized human foetal osteoblastic cell line (hFOB 1.19 cells; ATCC nr CRL-11372, Rockville, MD, USA) was cultured in 1:1 mixture of Ham’s F12 Medium and Dulbecco’s Modified Eagle’s Medium with 2.5 mM L-Glutamine (without phenol red; Thermo Fisher, Waltham, MA, USA). The medium was supplemented with 10 µg/mL of gentamicin and 0.25 µg/mL amphotericin B (Thermo Fisher, USA) and Foetal Bovine Serum (FBS; Thermo Fisher, USA) at the final concentration of 10%. Cells were cultured at 34 °C and 5% CO2 (according to ATCC culturing protocol for hFOB 1.19) in CB240 incubator (Binder, Tuttlingen, Germany). The above-mentioned cell culture medium was used in the experiments mentioned. hFOB 1.19 cell line was obtained from LGC Standards (Kiełpin, Poland).
3
Cell Viability and Cytotoxicity of Composites
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Prior to the experiment the composites were inserted into 24-well plates (Sarstedt, Nimbrecht, Germany) and immersed in 1 mL of culture medium for 1 h. Sterilized Raschig rings made of borosilicate glass 3.3 (Simax, Praha, Czech Republic) were placed on the top of the composites, followed by hFOB 1.19 cell seeding. In each well 5 × 104/cm2 hFOB 1.19 cells were seeded inside the Raschig ring.
Plates were incubated for 1 h at 34 °C and 5% CO2 in CB240 incubator (Binder, Germany) allowing the cells to attach to the composites. After 1 h, rings were removed, and the cells were incubated for 48 h. After that time the cell viability was estimated by measuring the mitochondrial activity (WST-1 test) and the cell cytotoxicity was measured by the leakage of lactate dehydrogenase (LDH cytotoxicity assay). All types of the composites were tested in at least 4 replicates.
Plates were incubated for 1 h at 34 °C and 5% CO2 in CB240 incubator (Binder, Germany) allowing the cells to attach to the composites. After 1 h, rings were removed, and the cells were incubated for 48 h. After that time the cell viability was estimated by measuring the mitochondrial activity (WST-1 test) and the cell cytotoxicity was measured by the leakage of lactate dehydrogenase (LDH cytotoxicity assay). All types of the composites were tested in at least 4 replicates.
4
Culturing Human Fetal Osteoblasts
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Human fetal osteoblasts (hFOB 1.19; ATCC CRL-11372, LGC Standards, Kiełpin, Poland) were cultured in a 1:1 mixture of Ham’s F12 Medium and Dulbecco’s Modified Eagle’s Medium (DMEM) with 2.5 mM L-Glutamine (without phenol red) supplemented with 10 µg/mL of gentamicin, 0.25 µg/mL amphotericin B, and Fetal Bovine Serum (FBS) at a final concentration of 10%. The cells were cultured at 34 °C and 5% CO2 in a CB240 incubator (Binder, Tuttlingen, Germany). The above-mentioned cell culture medium was used in further experiments. All reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). hFOB 1.19 cells were chosen for this study as a cell model due to their biological similarity to primary osteoblasts. The animal cell lines or human cancer cells were excluded from this research due to differences between the biology of animal cells, cancer cells, and the healthy human cells, such as hFOB 1.19.
5
Quantifying hFOB 1.19 Cell Proliferation
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The proliferation of hFOB 1.19 cells was evaluated using a WST-1 assay kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, the cells in a 24-well plate were incubated with the extract or fresh medium for 48 h, and then treated with 40 µL of WST-1 reagent, followed by 2 h incubation at 34 °C and 5% CO2 in a CB240 incubator (Binder, Tuttlingen, Germany)
The media were collected from each well, and transferred to 96-well flat bottom plate (Sarstedt, Nümbrecht, Germany). The optical density at 450 nm and 620 nm was measured using a plate reader, Epoch (BioTek Instruments, Winooski, VT, USA). The untreated cells and blank were included into each assay. The values of background and blank absorbance were subtracted from all the absorbance values. The percentage of proliferation was calculated by the equation below:
The control absorbance was calculated as a mean of triplicate.
The media were collected from each well, and transferred to 96-well flat bottom plate (Sarstedt, Nümbrecht, Germany). The optical density at 450 nm and 620 nm was measured using a plate reader, Epoch (BioTek Instruments, Winooski, VT, USA). The untreated cells and blank were included into each assay. The values of background and blank absorbance were subtracted from all the absorbance values. The percentage of proliferation was calculated by the equation below:
The control absorbance was calculated as a mean of triplicate.
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