U2OS cells were transfected with control or ALDOA siRNA as previously. 48 h post-transfection, 4000 cells were plated into wells of a 96-well plate (Corning). Cells were mock-treated or treated with the indicated doses of camptothecin (Sigma-Aldrich) or hydrogen peroxide (Sigma-Aldrich) as shown and incubated for 72 h. CellTiter-Glo 2.0 (Promega Corporation) was added to each well and luminescence was measured using a PHERAstar FSX detection system (BMG Labtech) and normalised back to the untreated wells27 (link).
Pherastar fsx detection system
Manufactured by BMG Labtech
Sourced in Germany
The PHERAstar FSX detection system is a high-performance multi-mode microplate reader designed for sensitive and accurate measurements in life science research. It offers a range of detection modes, including absorbance, fluorescence, and luminescence, allowing researchers to perform a variety of assays. The PHERAstar FSX provides reliable and reproducible results, making it a versatile tool for various applications in the laboratory.
Automatically generated - may contain errors
Lab products found in correlation
Camptothecin, by Merck Group (1 mentions)
Celltiter glo 2, by Promega (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
96 well plate, by Corning (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Forskolin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Optiplate, by PerkinElmer (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
4 protocols using pherastar fsx detection system
1
Evaluating Cell Viability under Stress
2
Cell Viability Assay with CellTiter-Glo 2.0
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CellTiter-Glo 2.0 Luminescent assay (Promega Corporation) was used to determine cell viability for dose response assays. Cells at a density of 500 cells/well in white walled 384-well plates were drug treated 24 h following seeding. Following 48 h of treatment, CellTiter-Glo 2.0 was added to each well according to the manufacturer’s instructions. Luminescence was recorded using a PHERAstar FSX detection system (BMG Labtech). Following background value removal, data was normalised to untreated controls. Dose response curves were generated and drug potency values (IC50) calculated using the GraphPad Prism 8 software.
3
Quantifying Cell Migration Mediated by CXCR4 and CCR7
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MDA-MB-231 and T47D cells were co-transfected with pCXCR4-H and pCCR7-M iDimerize plasmids. 48 hrs post-transfection cells were serum-starved for at least three h and then treated or untreated with A/C heterodimerizer (100mM final concentration) for 1 hour at 37 °C. 5 × 104 cells were then aliquoted in duplicate into Matrigel (BD, Franklin Lakes, NJ, USA) pre-coated 96 transwell assay plates with 5μm pore permeable support inserts (Corning, Somerville, MA, USA). Cells were then treated with CXCL12 at 10 ng/mL and/or CCL19 at 20 ng/mL final concentrations and allowed to migrate for 24 h at 37 °C. Cells were then detached from the underside of the transwell insert with 0.1% trypsin (Thermo Fisher Scientific, Waltham, MA, USA), loaded with Calcein AM/1 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and cell density was estimated by PHERAstar® FSX detection system (BMG Labtech, Ortenberg, Germany) relative to total cell input.
4
Measuring Intracellular cAMP Levels
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cAMP levels were assessed using the AlphaScreen Detection Kit (Perkin Elmer, Waltham, MA, USA). All cells were serum-starved for at least three hrs prior to the analysis. Briefly, cells were resuspended at 5 × 105 cells/ml in AlphaScreen stimulation buffer containing HBSS (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.5 mM IBMX, 5 mM HEPES, 0.1% BSA (all from Thermo Fisher Scientific, Waltham, MA, USA), pH 7.4 in the presence or absence of forskolin (10 μM final concentration) (Sigma Aldrich, St. Louis, MO, USA) and treated or untreated with A/C heterodimerizer (100 mM final concentration). 3000 cells/well were aliquoted in triplicate into 384-well OptiPlate (Perkin Elmer, Waltham, MA, USA) and stimulated with CXCL12 and CCL19 chemokine ligands at indicated concentrations for 15 min at 37 °C. Further, cells were processed according to the manufacturer’s protocol. Alpha Screen signal was recorded using PHERAstar® FSX detection system (BMG Labtech, Ortenberg, Germany) using the AlphaScreen optical module (Ex. 680 nm/Em. 520−620 nm). Data analysis was performed using GraphPrism software (San Diego, CA, USA).
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