For the experiments, three different colorectal cancer cell lines were used—HCA-2 (Duke C), LS 174T (Duke B), and SW 1116 (Duke A), along with a normal epithelial cell line known as CCD 841 CoN. All the cell lines were provided by ATCC (American Type Culture Collection ATCC®, Old Town Manassas, VA, USA). To promote optimal cell growth, we utilized specific culture media for each cell line. For CCD 841CoN and LS 174T cell lines, we used Eagle’s minimum essential medium (EMEM) (ATCC 30-2003), while Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM) from Sigma-Aldrich D8437 was used for the SW 1116 and HCA-2 cell lines. Additionally, both media were supplemented with 10% fetal bovine serum ((FBS), ATCC 30-2020), and 1% penicillin-streptomycin-neomycin stabilized solution (Sigma-Aldrich P4083) for optimal growth conditions.
P4083
Manufactured by Merck Group
Sourced in Switzerland
P4083 is a lab equipment product manufactured by Merck Group. It is a device used for precise temperature control and monitoring in various laboratory applications. The core function of P4083 is to maintain consistent and accurate temperatures within a specified range.
Automatically generated - may contain errors
Lab products found in correlation
Sw1116, by American Type Culture Collection (2 mentions)
Hca 2, by American Type Culture Collection (2 mentions)
Ls174t, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Ccl 2, by American Type Culture Collection (1 mentions)
Tcm 400, by Labomed (1 mentions)
D8437, by Merck Group (1 mentions)
Ls174t, by Merck Group (1 mentions)
Ccd841 con, by Merck Group (1 mentions)
A8960, by Merck Group (1 mentions)
D1530, by Merck Group (1 mentions)
Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions)
F9665, by Merck Group (1 mentions)
F0291, by Merck Group (1 mentions)
G9422, by Merck Group (1 mentions)
D4902, by Merck Group (1 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
Rocephin, by Roche (1 mentions)
6 protocols using p4083
1
Colorectal Cancer Cell Culture Conditions
2
Iron Stress Response in Cell Culture
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Culture was pretreated with 0.1% (v/v) antibiotics (penicillin 5units ml−1, streptomycin 5 μg ml−1, and neomycin 10 μg ml−1) (Sigma Aldrich, P4083-100 ml) for 24 h under the standard growth condition. This was transferred into 15-ml sterile tubes and centrifuged at 12,750 m s−2 for five seconds to collect cell pellets. The supernatant was removed by inversion, and cell pellets were washed three times with SW. Then, cells were re-suspended with SW + L1 containing the target total iron concentrations, [Fe]T = 0, 10, 100, 1000, and 10,000 nM. Cultures were maintained under the standard growth condition and monitored for the cell count using microscopy every 4–6 days for two consecutive subcultures. All data were collected triplicate.
3
HeLa Cell Cultivation and Analysis
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We used a HeLa cell line that was obtained several years ago for human cervical adenocarcinoma (CCL-2™; ATCC). HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM D6429; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (12103C; Merck-Millipore), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (P4083; Sigma-Aldrich) at 37°C, 5% CO2 in a humidified atmosphere. Cells (5 × 104 cells/well) were cultivated in 96-well plates [19 (link)]. The cells were observed, and images were acquired (magnification, 20×) using an inverted microscope (Labomed TCM 400) and then separated by centrifugation at 600 × g for 6 min. The supernatant was gently removed, and the cell pellet was counted. Three replicates were used for each treatment.
4
Colorectal Cancer Cell Lines Cultivation
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Three colorectal cancer cell lines were used in the experiments: HCA-2 (Duke C), LS 174T (Duke B), and SW1116 (Duke A), together with a normal cell line, CCD 841 CoN. All cell lines were supplied by ATCC (American Type Culture Collection ATCC®, Old Town Manassas, VA, USA). To ensure optimal conditions for cell growth, appropriate culture media were used: Eagle’s minimum essential medium (EMEM) (ATCC 30-2003) for CCD 841CoN and LS 174T cell lines, and Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM), Sigma-Aldrich D8437). For SW1116 and HCA-2 cell lines, both media were supplemented with 10% foetal bovine serum ((FBS), ATCC 30-2020) and 1% penicillin–streptomycin–neomycin stabilised solution (Sigma-Aldrich P4083).
5
Measuring Metabolic Activity through Oxygen Uptake
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Oxygen uptake was measured using a manometric respirometer (OxiTop Control 12 system, WTW, Xylem Analytics, Germany) (Fig. 1 ), as a non-invasive determinant of metabolic activity in the cells [23 (link), 24 (link)]. Each species was grown in batch cultures for 1–2 weeks and their oxygen uptake was continuously (56 min intervals) measured. Experiments were set up using aseptic techniques as follows: 0.5 mL (0.5 g DW (Dry Weight) L− 1) of culture was added to 80 mL of the WS in 1 L bottles. An antibiotic mix was added (P4083 Sigma-Aldrich) to suppress bacterial growth and ensure oxygen uptake was due only to the strains under investigation. Positive controls were set up similarly in 80 mL of YMB. Negative controls without fungi or yeast were set up for each WS. All cultures were carried out in replicates of four and incubated in the dark on a magnetic stirring platform at 120 rpm at room temperature (23–28 °C).
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Oxygen uptake measurements using the OxiTop system
6
Isolation and Osteogenic Differentiation of Human Bone Progenitor Cells
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Human bone progenitor cells (HBCs) were isolated from pieces of trabecular bone prepared from human bone marrow samples of patients undergoing orthopaedic surgery as described previously.35 (link) Informed consent was obtained in accordance with the local ethics committee (EKSG 08/14). The cells were culture expanded in proliferation medium (α-MEM, 22561021; Thermo Fisher Life Technologies, Switzerland) supplemented with 10% fetal calf serum (FCS) (F-9665; Sigma-Aldrich, Switzerland), 1% penicillin/streptomycin/neomycin solution (P-4083, Sigma-Aldrich) and 20 µL fibroblast growth factor 2 (FGF-2) (F-0291; Sigma-Aldrich) at 37°C in a humidified 5% CO2 atmosphere. The medium was exchanged every three to four days until cells reached approximately 90% confluence. Cells were harvested, resuspended in osteogenic differentiation medium (α-MEM, 10% FCS, 1% PSN, 44 µM L-ascorbic acid 2-phosphate (A-8960, Sigma-Aldrich), 2 mM β-glycerophosphate (G-9422, Sigma-Aldrich), 10 nM 1,25 dihydroxyvitamine D3 (D-1530, Sigma-Aldrich), and 10 nM dexamethasone (D-4902, Sigma-Aldrich)), containing different concentrations of ceftriaxone (Rocephin, Roche, Switzerland) and seeded in multiwell plates as well as 35 mm glass bottom dishes (P35G-0.170-14-C, MatTek Cooperation, USA). Cells seeded in osteogenic medium and proliferation medium without ceftriaxone supplementation served as control.
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