reason, we always attach the ring to the microdissection chamber first and only after asserting that it does not leak, we seed
the cells. It is important to minimize bubbles and wrinkles
when setting the ring in the microdissection chamber.
8. Do not disturb the cells by moving the dish after seeding. Leave
the cells in the incubator so that they can attach to the membrane and make protrusions. The time of adhesion and formation of cellular protrusions depends on both the cell types used
and the types of cellular protrusions of interest. In our case, we
incubate CAD cells for 2–3 h, when looking for filopodia
and/or TNTs but waited up to 10 days with media change
every 2–3 days for axons and dendrites in order to allow for cell
differentiation upon serum starvation. The time must be predetermined by each investigator based on their cell/protrusion
types.
9. Many different ways to induce cell protrusions have been published. Test different substances or physical conditions (i.e.,
hypoxia) that can work with your cell type to increase the
number of cellular protrusions you are interested
in. Remember that for each treatment, whole cell control
samples should be acquired.
10. The proper stimuli and/or concentrations should be predetermined by the investigator based on the cell type used and
cellular protrusions of interest. For instance, prolong H 2 O 2
exposure and/or high concentration can be harmful to the
cells and could result in the cells detaching from the membrane. In addition, to avoid disturbing the cells, care should be
taken when adding and aspirating solutions.
11. Time of cross-linking is critical. Proceed to the next step
immediately to avoid over-cross-linking.
12. All the procedures with the LCM must be done with filtered
PBS in the dish to avoid protein degradation [26].
13. Because time degrades proteins, longer periods of cutting must
be avoided (see Fig. 1d).
14. If your cuts are limited to a specific area, it helps if you outline
with a marker on the bottom of the dissection chamber the
desired area.
15. Both protrusion and membrane samples can be stored at
À80
C for several weeks.
16. Silver stain is not compatible with some types of MS.
17. Since the samples are in small volumes, the caps of the centrifuge tubes should be tightly closed and surrounded with parafilm to avoid evaporation and drying of the samples.
42
Ana Gordon and Karine Gousset
the cells. It is important to minimize bubbles and wrinkles
when setting the ring in the microdissection chamber.
8. Do not disturb the cells by moving the dish after seeding. Leave
the cells in the incubator so that they can attach to the membrane and make protrusions. The time of adhesion and formation of cellular protrusions depends on both the cell types used
and the types of cellular protrusions of interest. In our case, we
incubate CAD cells for 2–3 h, when looking for filopodia
and/or TNTs but waited up to 10 days with media change
every 2–3 days for axons and dendrites in order to allow for cell
differentiation upon serum starvation. The time must be predetermined by each investigator based on their cell/protrusion
types.
9. Many different ways to induce cell protrusions have been published. Test different substances or physical conditions (i.e.,
hypoxia) that can work with your cell type to increase the
number of cellular protrusions you are interested
in. Remember that for each treatment, whole cell control
samples should be acquired.
10. The proper stimuli and/or concentrations should be predetermined by the investigator based on the cell type used and
cellular protrusions of interest. For instance, prolong H 2 O 2
exposure and/or high concentration can be harmful to the
cells and could result in the cells detaching from the membrane. In addition, to avoid disturbing the cells, care should be
taken when adding and aspirating solutions.
11. Time of cross-linking is critical. Proceed to the next step
immediately to avoid over-cross-linking.
12. All the procedures with the LCM must be done with filtered
PBS in the dish to avoid protein degradation [26].
13. Because time degrades proteins, longer periods of cutting must
be avoided (see Fig. 1d).
14. If your cuts are limited to a specific area, it helps if you outline
with a marker on the bottom of the dissection chamber the
desired area.
15. Both protrusion and membrane samples can be stored at
À80
C for several weeks.
16. Silver stain is not compatible with some types of MS.
17. Since the samples are in small volumes, the caps of the centrifuge tubes should be tightly closed and surrounded with parafilm to avoid evaporation and drying of the samples.
42
Ana Gordon and Karine Gousset