Cells were cultured at a density of 105 cells/cm2 in RPE maintenance medium. On the day of the assay cell medium was removed and 100 μl of pHrodo™ BioParticles® (Molecular Probes) conjugate was added to the 96 well plate. Cell density was scaled at 106 cells for 1 vial of 100 μl of the bead conjugate. The cells were then incubated at 37 °C for 2 h, 6 h and 24 h. After incubation, the cells were washed three times with PBS to remove the undigested beads. Cells were then fixed with 4% paraformaldehyde for 15 min followed by three washes with PBS. For imaging the cells were counterstained using DAPI and observed with an inverted fluorescence microscope (Leica, Wetzlar, Germany). The average fluorescent intensity per cell was measured on SpectaMax Plus 384 microplate reader (Molecular devices, Sunnyvale, CA) at 509/533 nm. The average fluorescence value of control wells with no beads is subtracted from wells containing beads at the end of the assay to yield a cell-specific, net phagocytosis signal. Each assay was repeated three times.
Spectamax plus 384
Manufactured by Molecular Devices
Sourced in United States
The SpectaMax Plus 384 is a high-performance microplate spectrophotometer designed for accurate and efficient absorbance measurements. It features a 384-well microplate format and can perform measurements across a wide range of wavelengths.
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5 protocols using spectamax plus 384
1
Phagocytosis Assay with pHrodo Beads
2
Quantifying Secreted IL-1β in HT29 Cells
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Secreted protein levels of IL-1β from supernatants of HT29 cells were quantified by ELISA using the human IL-1β ELISA KIT (MyBioSource, San Diego, CA, USA) following manufacturer’s instructions. Briefly, in order to remove the cell debris, cell supernatants were centrifuged at 4 °C for 20 min at 1000 rcf and diluted 1:10 with PBS. Samples were incubated during 90 min at 37 °C in the precoated 96-well strip plate. Then, Detection Solution A was added during 45 min at 37 °C and after that, Detection Solution B was incubated for 45 min at 37 °C. TMB Substrate Solution was then added at 37 °C during 15 min. Finally, Stop Solution was added and absorbance was measured at 450 nm with the microplate reader SpectaMax Plus 384 (Molecular Devices, San Jose, CA, USA).
3
Phagocytosis Assay with pHrodo Beads
4
Xanthine Oxidase Inhibition Assay
5
Measuring HOBs Proliferation In Vitro
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The proliferation of HOBs in vitro was measured using MTT and BrdU test following the 12 and 24 hours of incubation according to the manufacturer guidelines. The measurement presented proliferation differences between the Au–TiO2, TiO2, and Ti (control) with a statistical significance level of 0.05 at 95% confidence level.
Cell proliferation was assessed by using the MTT Cell Proliferation Kit (#11465007001, Roche Diagnostics, Mannheim, Germany). 96-well microtiter plates with 5 × 103 cells per well were incubated for 12 and 24 hours and a sample of 100 μl eluate was obtained and cell proliferation was measured. The optical density of the samples was measured photometrically at 450 nm wavelength.
Proliferating cells were determined by using the BrdU (Bromdesoxyuridin) Cell Proliferation ELISA (enzyme-linked immunoadsorption) kit (Roche Diagnostics, Mannheim, Germany). 96-well microtiter plates with 5 × 103 cells per well were incubated and a sample of 150 μl eluate was obtained. The optical density of the individual samples was measured in a microplate reader (Specta Max plus 384, Molecular Devices, Sunnyvale, USA) at 450 nm wavelength.
Cell proliferation was assessed by using the MTT Cell Proliferation Kit (#11465007001, Roche Diagnostics, Mannheim, Germany). 96-well microtiter plates with 5 × 103 cells per well were incubated for 12 and 24 hours and a sample of 100 μl eluate was obtained and cell proliferation was measured. The optical density of the samples was measured photometrically at 450 nm wavelength.
Proliferating cells were determined by using the BrdU (Bromdesoxyuridin) Cell Proliferation ELISA (enzyme-linked immunoadsorption) kit (Roche Diagnostics, Mannheim, Germany). 96-well microtiter plates with 5 × 103 cells per well were incubated and a sample of 150 μl eluate was obtained. The optical density of the individual samples was measured in a microplate reader (Specta Max plus 384, Molecular Devices, Sunnyvale, USA) at 450 nm wavelength.
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