Computational protocol: Hydroxy-Terminated Conjugated Polymer Nanoparticles Have Near-Unity Bright Fraction and Reveal Cholesterol-Dependence of IGF1R Nanodomains

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Protocol publication

[…] UV–vis absorption and fluorescence spectra were measured at 25 °C in PBS using a Spectramax M5 plate reader (Molecular Devices) and SoftMax Pro software. A clear flat-bottom 96-well plate from Fisher Scientific was used and both UV and fluorescence spectra were measured from the bottom of the plate. All spectra were measured on triplicate samples and were corrected for the intrinsic absorption and fluorescence from PBS. Absorption spectra were measured from 350 to 750 nm with 5 nm intervals. Atto647N amine dye was excited at 580 nm to acquire the emission spectrum. Fluorescence of Pdots and Pdot–dye conjugate was monitored from 510 to 750 nm, after 470 nm excitation.The fluorescence quantum yield of Pdots was measured by comparison to a dilute solution (A460 = 0.09) of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM, Sigma-Aldrich) in methanol, using the published DCM quantum yield of 0.43 as a standard. Solutions of Pdots in PBS and dye in methanol were optically matched at 460 nm, the excitation wavelength. Both absorption and fluorescence spectra of Pdots and dye were taken under identical measurement conditions. The integrated intensities of fluorescence spectra and corrected refractive index were used to calculate the quantum yield using the equation: QYPdot(PBS) = QYDCM(methanol) × (absorbanceDCM/absorbancePdot) × (integrated areaPdot/integrated areaDCM) × (ηPdot PBS)2/(ηDCM methanol)2. Our quantum yield was consistent with literature values for conjugated polymer nanoparticles. [...] A glass coverslip (Fisher Scientific, coverslip no. 1 glass 22 mm × 22 mm) was cleaned with 1 M HCl for 2 h and washed thoroughly with Milli-Q water. Then the glass coverslip was dried using nitrogen flow. Purified Pdot-Atto647N conjugate at ∼0.5 μM was diluted 5000 times with PBS and pipetted onto a cleaned glass coverslip. After 10 min incubation, the coverslip was washed with 500 μL of PBS and imaged in PBS using a fluorescent microscope as above. A z-stack of 21 images was taken with 0.5 μm spacing, to avoid any impact from chromatic aberration. Stacked images were merged to an individual image and processed with the “smoothening” function in ImageJ. Then the image was mean-filtered with radius of 2 pixels and the brightness was adjusted in ImageJ. We analyzed 805 fluorescent spots for carboxy-Pdots and 842 spots for hydroxy-Pdots. The number of fluorescent puncta from Pdots was divided by the number of Atto647N puncta to give the bright fraction. The bright fraction plus the dark fraction equals 100%. […]

Pipeline specifications

Software tools SoftMax Pro, ImageJ
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Breast Neoplasms, Heart Diseases, Neoplasms
Chemicals Cholesterol, Imidazoles, Polyethylene Glycols