sEV isolated from the CM were quantified using a NanoOrange Protein Quantification Kit (LIFE TECHNOLOGIES). Briefly, sEV were diluted 1:100 in the 1× NanoOrange working solution and incubated at 90 °C for 10 min. Then, the samples were cooled for at least 20 min and the fluorescence was read at 470ex–570em nm wavelength, using a Spectra Max Gemini plate fluorometer (MOLECULAR DEVICES, Sunnyvale, CA). To characterize sEV surface epitopes, 5 μg of sEV were analyzed using the MACSPlex Exosome Kit (MILTENYI BIOTEC) that allows the detection of 37 surface markers and two isotype controls. sEV were incubated with MACSPlex Exosome Capture Beads and with MACSPlex Exosome Detection Reagent CD9, CD63, and CD81 for 1 h at RT. Then, 1 ml of MACSPlex Buffer was added to each sEV containing tube, and left for 15 min at RT. The sEV bound to the Capture Beads were washed by centrifuging at 3000×g for 5 min, and then resuspended in 150μL of MACSPlex Buffer. Samples were analyzed with a FACS Canto II (BD BIOSCIENCES). Surface markers were calculated subtracting the median signal intensity of each bead of the control sample from the signal intensities of the respective beads incubated with sEV.
Spectra max gemini plate fluorometer
Manufactured by Molecular Devices
The SpectraMax Gemini plate fluorometer is a multi-mode microplate reader that measures fluorescence intensity. It is designed to provide accurate and reliable fluorescence data for a variety of assay types. The instrument features a xenon flash lamp as the light source and can detect a wide range of fluorescent dyes and proteins.
Automatically generated - may contain errors
4 protocols using spectra max gemini plate fluorometer
1
Quantification and Characterization of Small Extracellular Vesicles
2
Protein Quantification of LoVo Spheroids
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At 10 days spheroids were harvested, washed twice in Phosphate Buffered Saline (PBS) and centrifuged at 6000 rpm for 5 minutes. LoVo spheroids were lysed in a buffer containing 20 mM TRIS–HCl (pH = 7.5), 1% SDS, 1 mM Na3VO4, 1 mM PMSF, 5% beta-mercaptoethanol and protease inhibitors. After sonication and centrifugation, proteins were quantified by NanoOrange (Thermo Fisher Scientific) methods according to manufacturer’s instructions. Briefly, samples were diluted in the NanoOrange working solution and incubated at 90°C for 10 minutes, protected from light. Successively, samples were cooled at room temperature for at least 20 minutes and transferred in a 96 wells plate. Fluorescence was read at 470ex–570em nm wavelength, using a Spectra Max Gemini plate fluorometer (Molecular Devices, Sunnyvale, CA). Protein concentration was determined using the reference standard curve (R2, coefficient of determination was reported in Fig 1 ).
3
Evaluating SPD's Impact on Chondrocyte Proliferation
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The effects of SPD addition were also evaluated on cell growth. To this end, 8 primary chondrocyte cultures were established from 8 dif ferent patients and their differential proliferative abilities were assessed as described in Ref. [41] by means of the PicoGreen dsDNA (Molecular Probes, Eugene, OR) quantitation reagent. 1000 cells were plated in triplicate wells of a 96 well plate either in plain medium (10% FCS DMEM) or with the addition of 100 nM SPD. Cells were left to proliferate and plates were collected at time 0, 3, 7, 10 and 14 days after plating. At each time the plates were emptied and stored frozen until analysis. 100 μl of cell lysis buffer (Molecular Probes) was added to each well and then combined with an equal volume of TE working solution containing the PicoGreen dsDNA quantitation reagent diluted 1:40. After 5 min incubation, the sample fluorescence was measured with a Spectra Max Gemini plate fluorometer (Molecular Devices, Sunnyvale, CA).
The instrument was set in the well scan mode with 480 excitation and 540 emission (cut off 515). Values were referred to fluorescence intensity at day 0 and expressed following the formula "percentage of growth increase = (fluorescence t n -fluorescence t 0 )/fluorescence t 0 *100".
The instrument was set in the well scan mode with 480 excitation and 540 emission (cut off 515). Values were referred to fluorescence intensity at day 0 and expressed following the formula "percentage of growth increase = (fluorescence t n -fluorescence t 0 )/fluorescence t 0 *100".
4
Quantification and Characterization of sEVs
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sEVs_IL-1 isolated from the CM were quantified using a NanoOrange Protein Quantification Kit (Life Technologies). Briefly, sEVs_IL-1 were diluted 1 : 100 in the 1x NanoOrange working solution and incubated for 10 minutes at 90 °C. The samples were then cooled for at least 20 minutes, and the fluorescence was read at 470ex–570em nm wavelength, using a Spectra Max Gemini plate fluorometer (Molecular Devices, Sunnyvale, CA). To characterize sEVs_IL-1 surface epitopes, 5 μg of sEVs_IL-1 were analyzed using the MACSPlex Exosome Kit (Miltenyi Biotec) that allows the detection of 37 surface markers and two isotype controls. sEVs_IL-1 were incubated with MACSPlex Exosome Capture Beads and with MACSPlex Exosome Detection Reagent CD9, CD63, and CD81 for 1 hour at room temperature (RT). Then, 1 ml of MACSPlex Buffer was added to each sEVs_IL-1 containing tube and left for 15 minutes at RT. The sEVs_IL-1 bound to the Capture Beads were washed by centrifuging for 5 minutes at 3000×g and then resuspended in 150 μL of MACSPlex Buffer. Samples were analyzed with a FACS Canto II (BD Biosciences). Surface markers were calculated subtracting the median signal intensity of each bead of the control sample from the signal intensities of the respective beads incubated with sEVs_IL-1.
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