Prior to encapsulation of Cy5.5, NPs were core-labeled with 10mM Alexa Fluor 488 maleimide (cytometry analysis) or Alexa Fluor 555 maleimide (bulk tissue homogenate analysis) purchased from Thermo Fisher for 4 h in pH 7.4 PBS, then cleaned of free dye by CL-6B size exclusion chromatography and concentrated back to the original concentration (40 mg/mL) using 30kDa spin filters (Millipore). Cy5.5-NPs were synthesized through encapsulation of Cy5.5 dye within the hydrophobic NP polymer core by mixing 5mg/mL Cy5.5 (DMSO) at 10%v/v with 40mg/mL PPS-NP (aqueous), then cleaned of free Cy5.5 dye using three successive 7kDa molecular weight cut-off Zeba spin desalting columns [16 (link)]. The resulting Cy5.5-NPs were injected i.d. into the mouse forelimb.
Protocol detail
Fluorescent Labeling and Encapsulation of Nanoparticles
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Alexa fluor 488
Alexa fluor 555
Bulk
Cy5 5
Dmso
Forelimb
Maleimide
Mouse
Np 10
Polymer
Size exclusion chromatography
Tissue
Corresponding Organization :
Other organizations : Georgia Institute of Technology, The Wallace H. Coulter Department of Biomedical Engineering
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Variable analysis
independent variables
- Encapsulation of Cy5.5 dye within the hydrophobic NP polymer core
- Fluorescence of Cy5.5-labeled NPs (cytometry analysis)
- Fluorescence of Cy5.5-labeled NPs (bulk tissue homogenate analysis)
- Concentration of PPS-NP (40 mg/mL)
- Concentration of Cy5.5 dye (5 mg/mL)
- Mixing ratio of Cy5.5 (DMSO) and PPS-NP (aqueous) at 10% v/v
- PH of PBS buffer (pH 7.4)
- Incubation time for labeling with Alexa Fluor 488 maleimide or Alexa Fluor 555 maleimide (4 h)
- Cleaning method for free dye removal (size exclusion chromatography and spin filters)
- Cleaning method for free Cy5.5 dye removal (Zeba spin desalting columns)
- Positive control: Alexa Fluor 488 maleimide-labeled NPs (cytometry analysis)
- Positive control: Alexa Fluor 555 maleimide-labeled NPs (bulk tissue homogenate analysis)
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