| Cell Line Species/Tissue: |
Acanthamoeba castellanii
/
Neff strain |
| Transfection Reagent: |
SuperFect |
Nucleic Acid:
|
DNA |
Growth Medium:
|
|
Percent Serum (%):
|
|
Reporter System:
|
|
Plasmid Purification Method:
|
Endofree Maxi prep |
Plate Format:
|
6-well in 3ml culture medium |
Number of Cells:
|
4x10e5 |
Percent Confluence(%):
|
|
Amount of Nucleic Acid (µg):
|
4 µl |
Amount of Enhancer (µl):
|
|
Amount of Reagent (µl):
|
20 µl |
Complex Incubation on Cells (hrs):
|
3 hrs |
Analysis Performed Post-Transfection (hrs):
|
24-48 hrs |
Transfection Efficiency (%):
|
|
| |
| Any modifications to the protocol?:
|
| |
| Notes: |
| Cells were cultured overnight at 25°C in a 6-well culture plate with 4x10e5 cells per well in 3 ml of culture medium. Four micrograms of plasmid DNA in 100 µl of amoeba culture medium were mixed with 20 ml of SuperFect (Qiagen), incubated for 10 minutes at room temperature and then diluted with 600 ml of culture medium. Adherent amoeba in a 6-well plastic culture plate were washed once with room temperature PBS.
After removing most of PBS, DNA-Superfect mixture was added drop-wise to the cells. Cells were incubated at 25°C for 3 hours to allow uptake of DNA-Superfect complexes. Cells were washed once with PBS, resuspended in 3 ml of fresh growth medium and incubated at 25°C for 24-48 hours. |
| References: |
| Intracellular localization and dynamics of myosin-II and myosin-IC in live Acanthamoeba by transient transfection of EGFP fusion proteins
Hyun-Hee Kong1,* and Thomas D. Pollard2,‡
Journal of Cell Science 115, 4993-5002, 2002 |