4.1 Cell culture
The hASCs were isolated from human adipose tissue by collagenase digestion and culture expansion in DMEM/F12 Ham's Medium, 10% FBS, 1% antibiotic/antimycotic according to published methods . Isolation of adipose-derived adult stem cells from human liposuction aspirates and surgically excised adipose depots was approved by Pennington Biomedical Reearch Center, Louisiana State University system with IRB number 23040.
4.2 Buffers and polymers
OptiMEM (Buffer O) is a commercial buffer that was purchased from Invitrogen (Carlsbad, CA) and stored at 4°C. Pulsing buffer (Buffer P), consisting of 125 mM KCl, 15 mM NaCl, 3 mM glucose, 25 mM HEPES, and 1.2 mM MgCl2 (pH7.4), is made from a 10× homemade stock solution, filtered, and stored at 4°C. After preparation, the solution is filtered through a 0.2-μm filter and stored at room temperature.
LMA1 stock was made to a 10% (wt/vol) of Poly-L-glutamic acid (mw 15-50 kDa, Sigma-Aldrich, St Louis, MO) in MQ-H2O. LME1 (polyethylene glycol, mw 8 kDa, Amresco, Solon, OH), LMP8 (poloxamer 188, mw 8 kDa, Spectrum Chemicals, New Brunswick, NJ), LMP1 (poloxamer 181, mw 2 kDa, Spectrum Chemicals), LMP5 (poloxamer 95, mw 1.8 kDa, Sigma-Aldrich), and LMV1 (polyvinylpyrrolidone, mw 40 kDa, Fisher Scientific, Pittsburg, PA) stocks were all made to 20% (wt/vol) in MQ-H2O. LMP3 (Pop313, donated from Expression Genetics, Huntsville, AL) was made to a stock concentration of 0.01% (wt/vol) in MQ-H2O. All stock buffers were stored at 4°C and were kept for 1 month.
4.3 Electroporation of plasmid DNA
Cells were pelleted and resuspended in 100 μL transfection buffer at a concentration of 1.0 × 106 cells/100 μL and then transferred to a sterile 3-mm Amaxa nucleofection cuvette. Cells were incubated with 2 μg reporter gene encoding plasmid DNA. The reporter genes, GFP (green florescence protein) and luciferase, were driven by CMV promoter at the 5'-end and terminated by the human growth hormone polyadenylation signal at the 3' end. The size of GFP and luciferase plasmid DNA are 4.7 and 4.3 kb, respectly. Cells were electroporated with use of the appropriate nucleofection program. Nucleofected cells were then rinsed with 500 μL of sterile culture medium and transferred to the well of a sterile 12-well plate. Cells were incubated at 37°C for 24 hours before analysis.
4.4 Flow cytometry
For flow cytometry analysis, GFP plasmid DNA was transfected into the targeted cells using the indicated formulation. GFP positive cells (% of total cells) were detected using flow cytometry. Before flow cytometry was performed, cells were harvested by trypsinization and resuspended in 1000 μL of PBS. Some of the cell suspension (500 μL) was removed and fixed in PBS + 1.0% formaldehyde solution before analysis. Cells were measured by a BD FACSCalibur (BD Biosciences; San Diego, CA).
4.5 Luciferase and protein assays
After incubation, cells were lysed and assayed for luciferase activity (Cell lysis kit, Promega; Madison, WI; part# E4030). Briefly, cell pellets were rinsed with PBS and lysed with 200 μL of lysis reagent with use of the freeze-thaw method. Cell lysates were kept at 4°C during analysis and stored at -20°C when not in use. Next, 20 μL of cell lysates (after spining) were transferred to a 96-well plate. Luciferase activity was measured by using a Packard LumiCount (Perkin Elmer; Boston, MA). All luciferase activity was normalized to the protein with use of the following equation: [(luciferase activity in RLU) × 200]/(per μg protein).
Protein assays were conducted by using a commercially purchased BCA protein assay kit (Thermo Scientific; Rockford, IL; Product # 23227). Briefly, 4 μL of cell lysate prepared from luciferase assay was transferred to a clear 96-well plate. Protein levels were measured by a Packard SpectraCount (Perkin Elmer). Protein assay measurements were calibrated to a BSA standard with a maximum of 40 μg and analyzed by I-Smart 2.0 (Perkin Elmer).
4.6 Electron microscopy
5 × 104 hASCs were seeded onto 13-mm round Thermanox cover slips (Thermo Fisher Scientific, Rochester, NY) placed in a sterile 24-well plate. Cells were grown until they reach 40%-50% confluence. All growth medium was removed from the wells, and cells were incubated at room temperature for 10 minutes in one of the following solutions before processing for SEM: DMEM, Buffer O, Buffer P, and 1/2 saline nucleofector solution. Briefly, cells were fixed in 1.25% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate (CAC) buffer for 1 hour at room temperature. Cells were then washed in 0.1 M CAC buffer, treated with 1% osmium tetroxide in 0.1 M CAC, and washed again. Cells were then dehydrated in ethanol before platinum-coating.