counted by mixing a sample of the cell solution with Trypan
blue (dilution 1:1). Count the cells using a hemocytometer or
an automated cell counter. Calculate the volume for your
desired cell concentration (i.e., 200,000 cells for TNTs) to be
seeded in an MMI live chamber dish.
3.2 Differentiated
CAD Cells
In order to obtain axonal and dendritic protrusions, we differentiated CAD cells by serum starvation.
1. Seed 30,000 CAD cells in an MMI live chamber dish.
2. To differentiate these cells, they have to be grown in OPTIMEM without FBS for 10 days.
3. The rest of the protocol is the same as for CAD cells.
3.3 Preparation
of MMI Live
Chamber Dish
Optimal conditions for cell attachment will depend on the cell type
and should be predetermined. For CAD cells, we use fibronectin
coating, but there are other extracellular matrix protein coatings
available for different cell lines (see Note 6).
1. Remove the membrane ring from the 35-mm dish with regular
tweezers and transfer it to the adhesive area of the
UV-permeable microdissection chamber. Press carefully with
your fingers and, once attached, do not move the ring to
prevent wrinkles and/or breakages (see Note 7).
2. To confirm that the membrane ring is properly attached and
does not leak, add 1 mL of filtered PBS to the dish and wait for
at least 5 min. If no leaks are observed, proceed with the sample
preparation. If PBS is seen on the outside of the membrane
ring, discard the ring/chamber and start anew.
3. Prepare 300 μL of filtered PBS and 24 μL of
fibronectin (6 mM).
4. Remove the PBS, add the fibronectin solution, and make sure
that the fibronectin solution covers the entire surface of the
ring since the membrane is hydrophobic. Place the dish in the
37
C/5% CO 2 incubator for 20 min.
5. Wash three times with filtered PBS and two times with CAD
media.
6. Add the desired volume of cells (in our case, 200,000 cells for
TNT samples) and leave them in the incubator until ready for
fixation (see Note 8).
7. Several protocols can be applied to increase cellular protrusions
and should be predetermined based on your cell types and
cellular protrusion of interest. We have included two of them
(serum starvation for axons/dendrites or exposure to H 2 O 2 for
TNTs), but many other stimuli can be applied depending on
your research goals (see Note 9).
Proteome of Cellular Protrusions Identified by LCM/MS
31
blue (dilution 1:1). Count the cells using a hemocytometer or
an automated cell counter. Calculate the volume for your
desired cell concentration (i.e., 200,000 cells for TNTs) to be
seeded in an MMI live chamber dish.
3.2 Differentiated
CAD Cells
In order to obtain axonal and dendritic protrusions, we differentiated CAD cells by serum starvation.
1. Seed 30,000 CAD cells in an MMI live chamber dish.
2. To differentiate these cells, they have to be grown in OPTIMEM without FBS for 10 days.
3. The rest of the protocol is the same as for CAD cells.
3.3 Preparation
of MMI Live
Chamber Dish
Optimal conditions for cell attachment will depend on the cell type
and should be predetermined. For CAD cells, we use fibronectin
coating, but there are other extracellular matrix protein coatings
available for different cell lines (see Note 6).
1. Remove the membrane ring from the 35-mm dish with regular
tweezers and transfer it to the adhesive area of the
UV-permeable microdissection chamber. Press carefully with
your fingers and, once attached, do not move the ring to
prevent wrinkles and/or breakages (see Note 7).
2. To confirm that the membrane ring is properly attached and
does not leak, add 1 mL of filtered PBS to the dish and wait for
at least 5 min. If no leaks are observed, proceed with the sample
preparation. If PBS is seen on the outside of the membrane
ring, discard the ring/chamber and start anew.
3. Prepare 300 μL of filtered PBS and 24 μL of
fibronectin (6 mM).
4. Remove the PBS, add the fibronectin solution, and make sure
that the fibronectin solution covers the entire surface of the
ring since the membrane is hydrophobic. Place the dish in the
37
C/5% CO 2 incubator for 20 min.
5. Wash three times with filtered PBS and two times with CAD
media.
6. Add the desired volume of cells (in our case, 200,000 cells for
TNT samples) and leave them in the incubator until ready for
fixation (see Note 8).
7. Several protocols can be applied to increase cellular protrusions
and should be predetermined based on your cell types and
cellular protrusion of interest. We have included two of them
(serum starvation for axons/dendrites or exposure to H 2 O 2 for
TNTs), but many other stimuli can be applied depending on
your research goals (see Note 9).
Proteome of Cellular Protrusions Identified by LCM/MS
31