Fitc conjugated cholera toxin

The AuNPs accumulation inside cells was analysed by the reflection of the indicated laser on their surface, using LSM510 Confocor II (Zeiss, Oberkochen, Germany). Cell membrane was visualised using FITC-conjugated cholera toxin (Sigma, cat. no.: C1655). Cell compartments were labelled with Cy3-coupled anti-early endosome antigen 1 (EEA1) (GeneTex, Irvine, CA, USA, cat. no.: GTX109638) and Alexa Fluor 647^® coupled anti-lysosomal associated membrane protein 1 (LAMP1) antibodies (Santa Cruz Biotechnology, Heidelberg, Germany, cat. no.: sc-8099). After incubation with AuNPs at the indicated conditions, cells washed with PBS were incubated for 1 h at room temperature in Petri dishes pre-coated with 20 mM polylysine for 24 h.
For TEM, cells were gently washed with PBS and fixed with 1% v/v glutaraldehyde in PBS for 1 h at room temperature. They were then post-fixed in 1% v/v osmium tetroxide in PBS for 1 h, and processed by ethanol dehydration, followed by embedding in epoxy resin. Observations were performed on a Philips CM120 TEM microscope operated at 80 kV.

For nanoparticle uptake assays, 0.5 x 10⁵ cells/mL of BMDCs and BMDMs were seeded into a 4-well Lab-Tek chambered coverslip. After 24 h of growth, the cells were incubated with both Dil-labelled nanocarriers, cNLCs and nNLCs, for 24 h at 37°C with 5% CO₂. Nanocarrier accumulation inside cells was monitored by time-lapse microscopy using a spinning disk confocal microscope (Andromeda, TILL-FEI). The Dil-labelled nanocarriers were visualised using the lipophilic dye excitation wavelength of 514 nm while plasma membranes were labelled with FITC-conjugated cholera toxin (Sigma, C1655) and visualised at the excitation wavelength of 488 nm. After acquisition, the images were processed in Icy 2.0.3.0 software, and spectral deconvolution was performed using NIS 5.20.01 software.

Cellular lipid raft levels were estimated based on staining of GM1 with cholera toxin stain. The cells seeded on glass coverslips and cultured overnight were washed with PBS and then incubated with 4 μg/ml FITC-conjugated cholera toxin (C1655, Sigma) in the ice-cold RPMI medium for 30 min in the dark. Cells were washed three times with PBS and fixed for 15 min in 4% paraformaldehyde. Cell signals were quenched with 50 mM NH₄Cl and then counterstained with DAPI. The coverslips were mounted with Gelvatol containing DABCO and visualized by fluorescence microscopy. Cholera toxin staining was quantified using ImageJ software.