1. Microscopy Lab - Cell Preferences
Easily scalable transduction yielding high percentage of expressing cells
Cells that adhere to glass
Relatively flat cells with an interesting morphology
Ligand induced translocation of signaling molecules
Functional assays
Cells can be brought into a low basal signaling state
WEHI Cell PM

2. Similar Transfection Efficiencies Obtained for WEHI-231 and RAW264
Similar Transfection Efficiencies Obtained for WEHI-231 and RAW264.7 Cells
Electroporated and plated the next day
Lipid transfection on glass
YFP
Propidium Iodide
Lipid transfection on glass

3. Morphology

4. Nucleus
WEHI-231
J774A.1
Raw264.7

5. Nuclear membrane
WEHI-231
J774A.1
Raw264.7

6. Endoplasmic Reticulum
WEHI-231
J774A.1
Raw264.7

7. Plasma membrane
WEHI-231
J774A.1
Raw264.7

8. Microtubules
WEHI-231

9. Microtubules
Raw264.7

10. Translocation of AKT-PH domain

11. YFP-AKT-PH Translocates to Plasma Membrane in RAW264.7 Cells
100 nM C5a

12. AKT-PH Translocation is Rapid and Transient

13. AKT-PH Translocation Occurs Reproducibly
100 nM C5a

14. RAW264.7 Cells tend to spread out and have an interesting morphology
Summary
Both RAW and J774 cells adhere well to polylysine treated and untreated glass.
Less that 5% of J774 cells transfect with methods tried. About 20-25% of RAW264.7 cells transfect with lipid based reagents (similar to levels obtained with electroporation of WEHI-231 cells).
RAW Cells tend to spread out and have an interesting morphology
YFP-AKT-PH translocated to plasma membrane of serum-deprived RAW264.7 cells when stimulated with C5a.
Raw Cell-Tubulin

15. Acknowledgements Nancy O’Rourke Liz Gehrig Wei Sun Park
Molecular Biology Lab
Raw Cell-Tubulin