Journal: International Journal of Molecular Sciences

Article Title: K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

doi: 10.3390/ijms22020598

Figure Lengend Snippet: Adenoviral transduction efficiency of murine mesenchymal stromal cells (MSC) isolated from three mouse strains. ( A ) Microscopy of non-obese diabetic NOD-MSC, C57BL/6-MSC, and BALB/c-MSC, transduced with 250 transduction units (TU)/cell adenovirus for GFP expression: BF—Bright field and GFP—fluorescence microscopy. ( B – D ) Dose-dependent transduction of MSC derived from NOD, C57BL/6, and BALB/c strains. The cells were incubated with increasing doses of adenovirus ranging from 0–2500 TU/cell; after 48 h the GFP expressing cells and the cell death determined by propidium iodide (PI) incorporation were evaluated by flow cytometry. The dose-dependent curves are different for NOD-MSC ( B ), C57BL/6-MSC ( C ), and BALB/c-MSC ( D ).

Article Snippet: The mixture was incubated for 20 min at room temperature and then was added dropwise to the MSC; (ii) AdV + Lipofectamine: a mixture containing 2.5 × 10 7 adenoviral particles and 10 μL P3000 in 125 μL Opti-MEM was added over a dilution of 7.5 μL Lipofectamine Reagent in 125 μL Opti-MEM; the final mix was incubated for 20 min at RT and then was added dropwise to the MSC; (iii) AdV + Polybrene: 2.5 × 10 7 adenoviral particles were added in 250 μL DMEM containing 2.5 μL solution 10 mg/mL polybrene; the mixture was added dropwise to MSC; (iv) AdV + Cholesterol: 2.5 μL solution containing 0.4 mg/mL cholesterol (in ethanol) were mixed with 2.5 × 10 7 adenoviral particles diluted in 250 μL DMEM and incubated for 20 min at RT; then the mixture was added dropwise to the MSC; (v) AdV + Poly-L-Lysine: 2.5 μL solution containing 0.1 mg/mL poly-L-Lysine were mixed with 2.5 × 10 7 adenoviral particles in 250 μL DMEM, and then the mixture was incubated with MSC in 1 mL serum free medium for 90 min; after this, 100 μL FBS was added to the cells; (vi) AdV + TransFast: 21 μL TransFast in 100 μL DMEM were mixed with 2.5 × 10 7 adenoviral particles and incubated for 15 min at RT, then were added to the MSC; (vii) AdV + Viromer Red: 540 μL Viromer Buffer with 2.5 × 10 7 adenoviral particles were mixed with 2.5 μL Viromer Red in 57.6 μL Viromer Buffer and incubated 15 min at RT, then the mixture was added to the cells.

Techniques: Transduction, Isolation, Microscopy, Expressing, Fluorescence, Derivative Assay, Incubation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

doi: 10.3390/ijms22020598

Figure Lengend Snippet: Adenoviral transduction of murine mesenchymal stromal cells in the presence of different potential transduction boosters. To induce GFP expression, MSC were incubated with 250 TU/cell adenovirus alone (AdV only) or in the presence of the K2 Transfection System (K2TS) (AdV + K2), Lipofectamine 3000 (AdV + Lipofectamine), 10 μg/mL Polybrene (AdV + Polybrene), 2 μg/mL free cholesterol (AdV + Cholesterol), 1 μg/mL poly-L-Lysine (AdV + Poly-L-Lys), TransFast (AdV + TransFast), or Viromer Red (AdV + ViromerRed). In parallel, MSC were transfected with pAdTrack-CMV using K2TS (pAdTrack + K2). After 48 h, the GFP expression was observed by fluorescence microscopy ( A ), and the number of the GFP-expressing cells was determined by flow cytometry ( B ). The percentage of GFP-positive cells determined by flow cytometry is represented by green columns and that of the dead cells colored with propidium iodide (PI)—in red columns. As is revealed, the K2TS is the most efficient reagent for boosting the adenoviral transduction of MSC. Bars, 20 µm.

Article Snippet: The mixture was incubated for 20 min at room temperature and then was added dropwise to the MSC; (ii) AdV + Lipofectamine: a mixture containing 2.5 × 10 7 adenoviral particles and 10 μL P3000 in 125 μL Opti-MEM was added over a dilution of 7.5 μL Lipofectamine Reagent in 125 μL Opti-MEM; the final mix was incubated for 20 min at RT and then was added dropwise to the MSC; (iii) AdV + Polybrene: 2.5 × 10 7 adenoviral particles were added in 250 μL DMEM containing 2.5 μL solution 10 mg/mL polybrene; the mixture was added dropwise to MSC; (iv) AdV + Cholesterol: 2.5 μL solution containing 0.4 mg/mL cholesterol (in ethanol) were mixed with 2.5 × 10 7 adenoviral particles diluted in 250 μL DMEM and incubated for 20 min at RT; then the mixture was added dropwise to the MSC; (v) AdV + Poly-L-Lysine: 2.5 μL solution containing 0.1 mg/mL poly-L-Lysine were mixed with 2.5 × 10 7 adenoviral particles in 250 μL DMEM, and then the mixture was incubated with MSC in 1 mL serum free medium for 90 min; after this, 100 μL FBS was added to the cells; (vi) AdV + TransFast: 21 μL TransFast in 100 μL DMEM were mixed with 2.5 × 10 7 adenoviral particles and incubated for 15 min at RT, then were added to the MSC; (vii) AdV + Viromer Red: 540 μL Viromer Buffer with 2.5 × 10 7 adenoviral particles were mixed with 2.5 μL Viromer Red in 57.6 μL Viromer Buffer and incubated 15 min at RT, then the mixture was added to the cells.

Techniques: Transduction, Expressing, Incubation, Transfection, Fluorescence, Microscopy, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

doi: 10.3390/ijms22020598

Figure Lengend Snippet: K2 Transfection Reagent K2TR optimization for the adenoviral transduction of murine MSC. The efficacy of K2TR to increase the yield of the adenoviral transduction of MSC derived from NOD ( A ), C57BL/6 ( B ), and BALB/c ( C ) mice were determined as % of GFP-positive cells (green lines). The cell death was determined by PI incorporation and expressed as % of PI-positive cells (red lines). On the left side of each graph, the GFP-positive cells and the cell death for the untransduced cells (−AdV) and for the cells transduced with 250 TU/cell adenovirus alone (+AdV) were illustrated, linked by a dotted line. Adenoviral particles were complexed with various K2TR doses. MSC were incubated with 10 μL/mL K2 Multiplier (K2M) and then the complexes of adenovirus–K2TR were added to the cells. The percentage of GFP-positive cells (green lines) induced by transduction and the percentage of PI-positive cells (red lines) were determined 48 h after transduction, by flow cytometry.

Article Snippet: The mixture was incubated for 20 min at room temperature and then was added dropwise to the MSC; (ii) AdV + Lipofectamine: a mixture containing 2.5 × 10 7 adenoviral particles and 10 μL P3000 in 125 μL Opti-MEM was added over a dilution of 7.5 μL Lipofectamine Reagent in 125 μL Opti-MEM; the final mix was incubated for 20 min at RT and then was added dropwise to the MSC; (iii) AdV + Polybrene: 2.5 × 10 7 adenoviral particles were added in 250 μL DMEM containing 2.5 μL solution 10 mg/mL polybrene; the mixture was added dropwise to MSC; (iv) AdV + Cholesterol: 2.5 μL solution containing 0.4 mg/mL cholesterol (in ethanol) were mixed with 2.5 × 10 7 adenoviral particles diluted in 250 μL DMEM and incubated for 20 min at RT; then the mixture was added dropwise to the MSC; (v) AdV + Poly-L-Lysine: 2.5 μL solution containing 0.1 mg/mL poly-L-Lysine were mixed with 2.5 × 10 7 adenoviral particles in 250 μL DMEM, and then the mixture was incubated with MSC in 1 mL serum free medium for 90 min; after this, 100 μL FBS was added to the cells; (vi) AdV + TransFast: 21 μL TransFast in 100 μL DMEM were mixed with 2.5 × 10 7 adenoviral particles and incubated for 15 min at RT, then were added to the MSC; (vii) AdV + Viromer Red: 540 μL Viromer Buffer with 2.5 × 10 7 adenoviral particles were mixed with 2.5 μL Viromer Red in 57.6 μL Viromer Buffer and incubated 15 min at RT, then the mixture was added to the cells.

Techniques: Transfection, Transduction, Derivative Assay, Incubation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

doi: 10.3390/ijms22020598

Figure Lengend Snippet: The optimization of K2M for adenoviral transduction of murine MSC. The efficacy of K2M to increase the adenoviral transduction of MSC-derived from NOD ( A ), C57BL/6 ( B ), and BALB/c ( C ) mice were expressed as % of GFP-positive cells (green lines). The K2M induced cytotoxicity was indicated by the % of PI-positive, dead cells (red lines). To optimize the concentration of K2M needed for transduction, MSC were incubated with increasing K2M concentrations (up to 50 μL/mL) for 90 min. Then, the adenovirus–K2TR complexes (5 μL/mL K2TR and 250 TU/cell adenoviral particle) were added to the cells. The percentage of GFP-positive cells and PI-positive cells at 48 h after transduction was determined by flow cytometry. The same fractions were evidenced also for untransduced cells (−AdV) and NOD-, C57BL/6- and BALB/c-MSC transduced with the adenovirus alone (+AdV), which were linked by a dotted line in the left side of the graphs.

Article Snippet: The mixture was incubated for 20 min at room temperature and then was added dropwise to the MSC; (ii) AdV + Lipofectamine: a mixture containing 2.5 × 10 7 adenoviral particles and 10 μL P3000 in 125 μL Opti-MEM was added over a dilution of 7.5 μL Lipofectamine Reagent in 125 μL Opti-MEM; the final mix was incubated for 20 min at RT and then was added dropwise to the MSC; (iii) AdV + Polybrene: 2.5 × 10 7 adenoviral particles were added in 250 μL DMEM containing 2.5 μL solution 10 mg/mL polybrene; the mixture was added dropwise to MSC; (iv) AdV + Cholesterol: 2.5 μL solution containing 0.4 mg/mL cholesterol (in ethanol) were mixed with 2.5 × 10 7 adenoviral particles diluted in 250 μL DMEM and incubated for 20 min at RT; then the mixture was added dropwise to the MSC; (v) AdV + Poly-L-Lysine: 2.5 μL solution containing 0.1 mg/mL poly-L-Lysine were mixed with 2.5 × 10 7 adenoviral particles in 250 μL DMEM, and then the mixture was incubated with MSC in 1 mL serum free medium for 90 min; after this, 100 μL FBS was added to the cells; (vi) AdV + TransFast: 21 μL TransFast in 100 μL DMEM were mixed with 2.5 × 10 7 adenoviral particles and incubated for 15 min at RT, then were added to the MSC; (vii) AdV + Viromer Red: 540 μL Viromer Buffer with 2.5 × 10 7 adenoviral particles were mixed with 2.5 μL Viromer Red in 57.6 μL Viromer Buffer and incubated 15 min at RT, then the mixture was added to the cells.

Techniques: Transduction, Derivative Assay, Concentration Assay, Incubation, Flow Cytometry

Journal: Cell metabolism

Article Title: Exercise induces hippocampal BDNF through a PGC-1α/FNDC5 pathway

doi: 10.1016/j.cmet.2013.09.008

Figure Lengend Snippet: (A-C) Five week old male wild-type BALB/c mice were injected with GFP- or FNDC5-expressing adenoviral particles intravenously. Animals were sacrificed seven days later and (A) inguinal/subcutaneous fat pads (WAT=white adipose tissues), (B) hippocampus, and forebrain (C) were collected and mRNA was prepared and gene expression was assessed by qPCR. Data are shown as mRNA levels relative to Rsp18 expression, expressed as mean ± SEM. *P < 0.05 compared to wild type control group. (D) Model of the hippocampal PGC-1α/FNDC5/BDNF pathway in exercise. Endurance exercise stimulates increases hippocampal Fndc5 gene expression through a PGC-1α/Errα transcriptional complex. This elevated Fndc5 gene expression stimulates in turn Bdnf gene expression. BDNF is the master regulator of nerve cell survival, differentiation and plasticity in the brain. This will lead to improved cognitive function, learning and memory, which are known beneficial effects of exercise on the brain. See also Figure S4.

Article Snippet: Peripheral delivery of FNDC5 by adenoviral vectors High titer GFP- or FNDC5-expressing adenoviral particles were obtained by ViraQuest Inc. (North Liberty, IA).

Techniques: Injection, Expressing, Gene Expression, Control