9. Instead of leaving 200 μl of medium in the wells, you can also
aspirate all medium and add 200 μl of fresh medium supplemented in FCS/FBS. Adding 200 μl of medium supplemented
in 10% FCS/FBS and 200 μl of lipoplexes will result in 5%
FCS/FBS in the final transfection mixture. At this step, by
adding medium supplemented with higher content of
FCS/FBS, you can control the % of FCS/FBS in the transfection mixture, if needed. Also note that transfection efficiency
may vary depending on the procedure—it may be different
when you add fresh medium instead of leaving the medium in
wells.
10. Twenty-four hours is an optimal time to verify expression of
most reporter genes used to test efficiency of transfection like
those encoding EGFP or luciferase. This time may vary for
different reporter genes, plasmids, and cells from 24 to 96 h
after transfection.
11. For staining of live cells, dye that penetrate cell membrane like
Hoechst 33342 should be used. For example propidium iodide
is not cell permeable and requires cell permeabilization in the
staining procedure, so it cannot be used for the staining of live
cells.
12. Vaccine doses containing 62.5–125 μg DNA can be used for
chicken immunization and those containing 10 μg for mice
immunization. It is recommended to vaccinate animals twice—
chickens at seventh and 21st day of life while mice at 35th and
49th day of life.
13. It is recommended that all tested serum samples, positive and
negative controls should be analyzed in duplicate.
14. At all incubation steps, plates must be sealed with Parafilm to
prevent evaporation of the solvent.
15. In all washing steps, use an automated 96-well plate washer or
remove washes by flicking the plate over a sink followed by
patting the plate on a paper towel.
16. The examples of ELISA results of chicken and mice immunization with DNA vaccines against influenza virus are presented in
Stachyra et al. [9].
Acknowledgments
This chapter is dedicated to Professor Tadeusz Chojnacki of the
Institute of Biochemistry and Biophysics, Polish Academy of
Sciences (PAS) in Warsaw, the Father of Polish Polyprenology
who started the cooperation between the Institute of Biochemistry
and Biophysics PAS and Jagiellonian University. We would like to
thank Professor Ewa Swiezewska of the Institute of Biochemistry
402
Monika Rak et al.
aspirate all medium and add 200 μl of fresh medium supplemented in FCS/FBS. Adding 200 μl of medium supplemented
in 10% FCS/FBS and 200 μl of lipoplexes will result in 5%
FCS/FBS in the final transfection mixture. At this step, by
adding medium supplemented with higher content of
FCS/FBS, you can control the % of FCS/FBS in the transfection mixture, if needed. Also note that transfection efficiency
may vary depending on the procedure—it may be different
when you add fresh medium instead of leaving the medium in
wells.
10. Twenty-four hours is an optimal time to verify expression of
most reporter genes used to test efficiency of transfection like
those encoding EGFP or luciferase. This time may vary for
different reporter genes, plasmids, and cells from 24 to 96 h
after transfection.
11. For staining of live cells, dye that penetrate cell membrane like
Hoechst 33342 should be used. For example propidium iodide
is not cell permeable and requires cell permeabilization in the
staining procedure, so it cannot be used for the staining of live
cells.
12. Vaccine doses containing 62.5–125 μg DNA can be used for
chicken immunization and those containing 10 μg for mice
immunization. It is recommended to vaccinate animals twice—
chickens at seventh and 21st day of life while mice at 35th and
49th day of life.
13. It is recommended that all tested serum samples, positive and
negative controls should be analyzed in duplicate.
14. At all incubation steps, plates must be sealed with Parafilm to
prevent evaporation of the solvent.
15. In all washing steps, use an automated 96-well plate washer or
remove washes by flicking the plate over a sink followed by
patting the plate on a paper towel.
16. The examples of ELISA results of chicken and mice immunization with DNA vaccines against influenza virus are presented in
Stachyra et al. [9].
Acknowledgments
This chapter is dedicated to Professor Tadeusz Chojnacki of the
Institute of Biochemistry and Biophysics, Polish Academy of
Sciences (PAS) in Warsaw, the Father of Polish Polyprenology
who started the cooperation between the Institute of Biochemistry
and Biophysics PAS and Jagiellonian University. We would like to
thank Professor Ewa Swiezewska of the Institute of Biochemistry
402
Monika Rak et al.