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Anwendungsbericht: Transfektion von A7 retinalen Astrozyten

Transfektion von Astrozyten mit dem
K2® Transfection System

Dieser Anwendungsbericht beschreibt die Transfektion von A7 retinalen Astrozyten mit gWizGFP (green fluorescence protein) reporter plasmid und dem K2® Transfection System. Nach 48h beträgt die Transfektionseffizienz bis zu 40% bei einer Viabilität der Zellen von 70 bis 90%.

 

Transfection of an immortalized retinal cell line using

K2® transfection reagent – Brief Report

Ding Wen (Roger) Chen and Marianna Foldvari
School of Pharmacy and Waterloo Institute of Nanotechnology,
University of Waterloo, Waterloo, ON, Canada N2L 3G1

Original purpose of the experiment:

To determine the most optimal K2® formulation for the transfection of A7 retinal astrocytes seeded with different population by evaluating its transfection efficiency and toxicity after 48 hours.

 

Material:

  • K2® Transfection System (K2® Reagent + K2® Multiplier) (Cat#T060, Biontex, Germany)
  • gWiz GFP (green fluorescence protein) reporter plasmid (Aldevron, Fargo, ND, USA)
  • A7 rat astrocytes [1,2]
  • DMEM/High Glucose media (supplemented with 10% fetal bovine serum and 1% penicillin streptomycin) (Thermo Fisher Scientific Waltham, MA, USA)
  • TrypLE Express reagent (Thermo Fisher Scientific)
  • 12- or 24- well tissue-culture treated plates (MatTek Corporation, Ashland, MA, USA)
  • MitoTracker® Deep Red FM (Thermo Fisher Scientific)
  • CellMask™ Deep Red Plasma Membrane Dye (Thermo Fisher Scientific)

 

Method:

  1. A7 rat astrocyte, an immortalized astrocyte cell line was cultured with DMEM/High Glucose media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (complete media) in T75 cell culture treated flask until 70% confluency.
  2. Cells were trypsinized using TrypLE Express and appropriate number of cells (25,000, 50,000, or 100,000 cells) were seeded into each well of either 12-well (for 50,000 and 100,000 cell seeding density) or 24-well (for 25,000 cell seeding density) tissue culture treated plates supplemented with 1mL of complete media . The plates were incubated in a 37°C incubator with 5% CO2 overnight prior to transfection.
  3. Two hours prior to transfection, 3.75µL of K2® Multiplier were added directly into each well containing cells and the plates were incubated at 37°C.
  4. In the meantime, K2® transfection particles were prepared according to manufacturer’s protocol. Please refer to Table 1 for specific quantities used.
  5. After two hours of K2® Multiplier incubation, the prepared K2® transfection particle was added into each well and incubated at 37°C for 48 hours. The specific dose volume varies between the formulations and seeding density as shown in Table 1.
  6. After 48 hours of transfection, cells were trypsinized using TrypLe™ Express and stained with 1mL of 1µM MitoTracker® Deep Red for 30 minutes at 37°C prior to flow cytometry evaluation. Transfection efficiency was determined based on GFP expression and the viability was evaluated based on MitoTracker® fluorescent emission.

Table 1 – Composition of the different K2® formulations

K2-Astrozyten-Tabelle

 

Results:

 

A7 retinale Astrozyten transfiziert mit dem K2® Transfection System - Transfektionsrate

 

Figure 1 – Comparison between the transfection efficiency of A7 retinal astrocytes transfected with two different formulation ratios of K2® transfection reagents after 48 hours.

 

A7 retinale Astrozyten transfiziert mit dem K2® Transfection System - Viabilität

Figure 2 – Comparison between the viability of A7 retinal astrocytes transfected with two different formulation ratios of K2® transfection reagent after 48 hours.

 

A7 retinale Astrozyten transfiziert mit dem K2® Transfection System - Mikroskopische Bilder

Figure 3 – Confocal microscopic images of A7 astrocytes transfected with K2 transfection reagent (Formulation 2) after 48 hours. Green representing GFP expression and red representing the cellular membrane stained with CellMask™ Deep Red Plasma Membrane dye.

 

 

Conclusion:

 

K2® transfection system has demonstrated to be effective in the transfection of A7 cells and is currently being used in downstream experiments.

 

 

 

References

[1] H.M. Geller, M. Dubois-Dalcq, Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector. J Cell Biol. 107 (1988) 1977-1986.

[2] M. Noble, K. Murray, Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell. EMBO J.  3 (1984) 2243-2247.

[3] Chen DW and Foldvari M., In vitro bioassay model for screening non-viral neurotrophic factor gene delivery systems for glaucoma treatment. Drug Deliv. Transl. Res. (2016) doi:10.1007/s13346-016-0324-9