The quantitative analysis of secreted molecules in conditioned media (CM) of TRF2-compromised and TRF2 overexpressing HCT116 cells was performed by using the Bio-Plex multiplex system (Bio-Rad, Milan, Italy) based on xMAP technology. that makes use of magnetic beads coated with specific antibodies raised against target analytes. Microspheres are internally labeled with red and infrared fluorophores. Each bead is bound to a specific antibody, thus allowing the simultaneous detection of multiple analytes within one sample. Following reaction of coupled beads with target analytes, a biotinylated antibody is added for the detection, which is then finalized by adding phycoerytrin-conjugated streptavidin. All steps were performed according to manufacturer's instructions for determining the concentration of the indicated molecules. Data were acquired using a Bio-Plex MAGPIX Multiplex Reader system equipped with a Bio-Plex Manager software v. 6.1 (BioRad). All washing steps were performed on the Bio-Plex magnetic wash station (BioRad). Measurements were performed in triplicate on CM samples (50 μl) using the Bio-Plex Pro human cancer biomarker panel 2 (Cat. No. 171AC600M, BioRad), according to the manufacturer's protocol. Standard curves optimization and the calculation of analyte concentrations were performed by using the Bio-Plex Manager software.
Bio plex magnetic wash station
Manufactured by Bio-Rad
The Bio-Plex magnetic wash station is a laboratory equipment designed to automate the washing process for Bio-Plex assays. The device utilizes magnetic beads to capture and wash samples, ensuring consistent and efficient sample preparation for downstream analysis.
Automatically generated - may contain errors
3 protocols using bio plex magnetic wash station
1
Multiplex Analysis of Secreted Molecules
2
Cytokine Profiling of CBDA-Treated HaCat Cells
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HaCat cells were seeded at 3.0 × 105 cells per well in 6-well plates. After 24 h, cells were treated with CBDA at 2.5, 5, and 10 µM concentration levels, in free-serum medium, for 48 h exposure time. Following cell harvesting, supernatants were collected and stored at −80 °C until use. Cytokines, chemokines and growth factors levels were evaluated using a Bio-Plex MAGPIX Multiplex Reader system (Bio-Rad, Milan, Italy) equipped with a Bio-Plex Manager software v 6.1 (BioRad) according to manufacturer’s instructions [58 (link)]. All washing steps were performed on the Bio-Plex magnetic wash station (BioRad). Measurements were performed in triplicate on samples (50 µL) using Bio-Plex Pro™ Human Cytokine 27-plex Assay (Bio-Rad). Standard curves optimization and the calculation of analyte concentrations were performed by using the Bio-Plex Manager software. Data were expressed as mean ± SD.
3
Cytokine and Chemokine Profiling Protocol
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At the time of cell harvest, supernatants were also collected and stored at − 80 °C until use. Cytokine and chemokine production was evaluated by using a Bio-Plex MAGPIX Multiplex Reader system (Bio-Rad, Milan, Italy) equipped with a Bio-Plex Manager software v 6.1 (BioRad) according to manufacturer’s instructions [35 (link)]. All washing steps were performed on the Bio-Plex magnetic wash station (BioRad). Measurements were performed in triplicate on samples (50 µl) using the Bio-Plex Pro Human Cytokine 40-plex assay kit (Cat. No. 171AK99MR2, BioRad). Standard curves optimization and the calculation of analyte concentrations were performed by using the Bio-Plex Manager software. Data were expressed as mean ± SD.
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