MCF-7 cells (ATCC HTB-22) and MDA-MB-231 (ATCC HTB-26) human mammary carcinoma cells were incubated with PLP, LCL-PLP or liposomes as control (C). Cells were cultured in DMEM/F12 medium (Life Technologies Europe B.V., Bleiswijk, The Netherlands) containing 1.2 g/L sodium bicarbonate, 3.6 g/L HEPES, 3.2 g/L D-glucose, 2.5 mM L-glutamine, and supplemented with 10% FBS. 10 3 cells/well were plated in a 96-well plate for 24 h. Subsequently, liposomal PLP and free PLP or vehicle were added in the respective wells. The anti-proliferative effect was determined over 48 h, by ELISA BrdU-colorimetric immunoassay (Roche Applied Science, Penzberg, Germany) according to the manufacturer's instructions. This technique is based on the incorporation of the pyridine analogue bromodeoxyuridine (BrdU) instead of thymidine into the DNA of proliferating cells. To detect BrdU incorporated in newly synthesized cellular DNA, a monoclonal antibody conjugated with peroxidase was added. After 90 min of incubation, cell lysates were washed three times with PBS. The immune complexes were detected by adding the substrate of peroxidase (tetramethyl-benzidine). The reaction product was quantified by measuring the absorbance at 450 nm with a reference wavelength of 655 nm.
Elisa brdu colorimetric immunoassay
Manufactured by Roche
Sourced in Germany
The ELISA BrdU-colorimetric immunoassay is a laboratory instrument used to measure cell proliferation. It detects and quantifies the incorporation of the synthetic thymidine analog bromodeoxyuridine (BrdU) into the DNA of dividing cells. The assay is based on an ELISA (Enzyme-Linked Immunosorbent Assay) technique, where the amount of BrdU incorporated is determined colorimetrically.
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Lab products found in correlation
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Cell Signaling Technology (1 mentions)
Gm csf, by Cell Signaling Technology (1 mentions)
Ebm basal medium, by Lonza (1 mentions)
Egm 2 singlequot kit supplements and growth factors, by Lonza (1 mentions)
Huvecs, by Lonza (1 mentions)
5 protocols using elisa brdu colorimetric immunoassay
1
Anti-proliferative Effect of Liposomal PLP
2
Ajuga Extracts Impact on Melanoma and Colon Cancer
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To determine the effect of Ajuga sp. extracts on B16.F10 murine melanoma and C26 colon carcinoma cells proliferation, 5 × 103 cancer cells/well were cultured in 96-well plates for 24 h. The range of concentrations for each extract was selected based on previous studies regarding in vitro cytotoxic activity of Ajuga sp. (Sadati et al., 2012 (link)) and the effect was measured in triplicate samples for the controls (cells incubated in medium alone) and for each concentration of the vegetal extracts. To screen for ethanol toxicity, cells were incubated with the same concentrations of the solvent as those used for the preparation of the ethanolic extracts. The proliferative activity of the cancer cells after different treatments was tested using ELISA BrdU-colorimetric immunoassay (Roche Applied Science, Penzberg, Germany) as previously described (Licarete et al., 2017 (link)). Cell proliferation was calculated as percentage of untreated cells (control value). To measure the effectiveness of the treatments, the IC50 was calculated by GraphPad Prism version 6 for Windows software.
3
In Vitro Antiproliferative Effects of PEG-EV-DOX
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The in vitro antiproliferative effects of PEG-EV-DOX treatment was assessed on B16.F10 melanoma cells in monoculture as well as in co-culture with bone marrow differentiated M2 TAM with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF, Cell Signaling Technology, MA, USA) and 20 ng/ml interleukin-4 (IL-4, Cell Signaling Technology, MA, USA), as previously described by Rauca et al., 2018.87 (link) For this, ELISA BrdU-colorimetric immunoassay (Roche Applied Science, Penzberg, Germany) was used, as previously described and according to the manufacturer’s instructions.62 (link) Thus, to test the efficacy of PEG-EV-DOX as compared to free DOX, B16.F10 melanoma cells were seeded in a 96-well at a ratio of 5000 cells/well for monocultures, while for co-cultures a ratio of 4000 B16.F10 cells to 1000 M2 TAM/well was used. This cell density ratio (4:1) was reported to approximate the in vivo physiological conditions of murine melanoma development.88 (link) After cells were allowed to attach for 24 h, serial concentrations of PEG-EV-DOX or DOX (ranging between 0.008 and 0.125 µM DOX) were tested in triplicate to assess the IC50 values after 24 h incubation with the treatment. The results were expressed as % of proliferation compared to control (untreated cells in monoculture and, respectively, in co-culture).
4
Evaluating Liposome Cytotoxicity on Cancer and Normal Cells
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Murine 4T1 breast cancer cells and human FaDu squamous cell carcinoma cells were obtained from the ATCC. Cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL). Normal human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Basel, Switzerland) and cultured in EBM basal medium supplemented with EGM-2 SingleQuot kit supplements and growth factors (Lonza).
FaDu squamous cell carcinoma cells or 4T1 metastatic breast cancer cells were seeded at a density of 3,000 cells per well in a 96-well plate. HUVECs, which served as control (normal cells), were seeded at a density of 4,000 cells per well in a 96-well plate. After 24 hours, cells were treated with ω-liposomes and C-liposomes at the indicated concentrations for 24 hours. To determine the number of dividing cells, bromodeoxyuridine (BrdU) reagent was added to the cells for 4–6 hours and an ELISA BrdU colorimetric immunoassay (Hoffman-La Roche Ltd, Basel, Switzerland) was performed, according to the manufacturer’s protocol.
FaDu squamous cell carcinoma cells or 4T1 metastatic breast cancer cells were seeded at a density of 3,000 cells per well in a 96-well plate. HUVECs, which served as control (normal cells), were seeded at a density of 4,000 cells per well in a 96-well plate. After 24 hours, cells were treated with ω-liposomes and C-liposomes at the indicated concentrations for 24 hours. To determine the number of dividing cells, bromodeoxyuridine (BrdU) reagent was added to the cells for 4–6 hours and an ELISA BrdU colorimetric immunoassay (Hoffman-La Roche Ltd, Basel, Switzerland) was performed, according to the manufacturer’s protocol.
5
HSP90 Inhibitors Modulate Proliferation
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NCI-H295R (8 × 103 cells per well) and MUC-1 (6 × 103 cells perwell) cells were treated with HSP90 inhibitors (luminespib, ganetespib, 17-AAG, novobiocin and silibinin) for 12 and 48 h, respectively. BrdU assay was performed using cell proliferation ELISA (BrdU, colorimetric immunoassay) (Roche, Basel, Switzerland) for quantification following manufacturer's instructions.
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