Sabharwal, N., Holland, E.C., and Vazquez,M.
SUPPLEMENTAL INFORMATION
EXPERIMENTAL
1. Cell Culture
The development of GPCPDGF cells has been described in previous publications [4-6]. Briefly, primary cell culture was established from mice transgenic for the expression of the tv-a receptor for the retroviral vector RCAS, expressed from the Nestin promoter. These so-called Ntv-a primary cultures were then infected with a RCAS-PDGF vector to develop GPCPDGF cells used for this study [4]. GPCPDGF cells were cultured with sterile Dulbecco’s Minimum Essential Medium Eagle (DMEM, pH 7.1-7.4) (Mediatech Inc., catalog # 10-013-CV), supplemented with 2% L-Glutamine (Mediatech Inc., catalog # 25-005-CI), 2% Penicillin-Streptomycin-Amphotericin B solution 100x (Mediatech Inc., catalog # 30-004), and 10% fetal bovine serum (FBS) (Mediatech Inc., 35-010-CV). The cells were grown onto sterile polystyrene tissue culture flasks (BD Biosciences, catalog # 353136) and incubated at 37oC with 5% CO2.
2. Liposome Preparation
Liposomes were extruded for intracellular labeling of live cells as described in previous publications [9]. Briefly, lipid sheets were formed by dissolving 1-Oleoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine (OPPC, Avati Polar, catalog # 850475P) and 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine- chloride (DOPC+, Avati Polar, catalog # 890704P) in chloroform at 1:1 molar ratios. The solution was further vaccum dried for 3 h to obtain lipid sheets that could be stored at -20oC until used (<24 h). The lipid sheets were then hydrated using 2 ml of PBS containing 1nM of QDs and biotinylated antibodies. This 1 mg ml-1 liposome solution was then frozen (20 min.) and thawed three to five times to help promote liposome laminarity and prevent membranes from fouling [18]. Solutions were extruded through a polycarbonate membrane filter (Whatman, catalog # 800281) in order to obtain liposomes smaller than 200nm in diameter. Extrusion for more than 7 consecutive passes was also used in order to minimize liposome polydispersity within final suspensions [2].
3. Imaging
All images were acquired using bright field and confocal fluorescence microscopy (Leica TCS SP2 Confocal Scanning Microscope) with 63X oil immersion objective (NA 1.4). Light microscopy was performed using a Nikon TE 2000 inverted microscope (Morrell Instruments, Melville, NY) with a 20X dry objective (NA 0.40). Identical imaging conditions were used for each set of experiments.
4. MTT Assay
GPCPDGF cells were lipoinfected with liposomes having S-QDs and were grown at 1x106 cells ml-1 cell concentration on 6 well plates for 24 h as described above. These cells were then analyzed with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay kit (Sigma Aldrich, catalog # CGD1), a colorimetric measurement of mitochondrial activity of live cells [7]. The dye (purple formazan) instensity was determined spectrophotometrically by ELISA reader (Biotek, Synergy, TK). Results were reported as the absorbance at 570 nm minus background at 690 nm. The spectrophotometer analysis or absorbance indicates the number of mitochondria and hence the living number of cells in the sample. Viability was determined by comparison to control GPCPDGF cultures with no S-QDs lipofection.
Figure S1: Toxicity of T-QDs in GPCPDGF cells as assessed by MTT assay of lipofected ( ) and non-lipofected ( ) cultures.
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