3.4 Fixation
As previously stated, cellular protrusions are fragile and must be
fixed prior to LCM isolation [26, 27]. However, while a strong
fixative like glutaraldehyde is ideal for maintaining the integrity of
cellular protrusions, it is irreversible and does not allow for efficient
protein extraction post cell lysis (Fig. 1a, b). On the other hand, we
have recently demonstrated that using a cleavable crosslinker like
DTBP not only preserves cellular protrusions [26] but it also allows
for a greatly improved protein recovery after cell lysis (Fig. 1c).
Overall, the use of DTBP along with the alternate protein extraction protocol we have developed [26] allows for the reduction of
the overall number of cellular protrusions needed to be isolated by
LCM, as it greatly increases the number of spectra, number of
proteins, and number of total unique proteins identified by MS
compared to glutaraldehyde-fixed samples (Fig. 2).
1. To increase the number of cellular protrusions, a concentration
of 100 μM of H 2 O 2 in warm CAD media is added to the cells
for 5 min (see Note 10).
2. Add 100 μL for 500 μL total volume or 200 μL for 1 mL total
volume of the fixation solution 1 (4% PFA solution) directly to
the cells in their culture media and wait for 5 min.
3. Aspirate and add 1 mL of fixation solution 1 for 15 min (see
Note 11). Change fixative solutions by tilting the dish, allowing and carefully aspirating the solution near the wall, without
disturbing cells, or they might detach and cellular protrusions
could break. Take the same considerations for steps 4 and 5.
4. Aspirate and add 1 mL of fixation solution 2 (5 mM DTBP
solution) for 15 min.
5. Aspirate and add 1 mL of filtered PBS (see Note 12).
We recommend preestablishing the best conditions (time and
concentration) for induction of cellular protrusions with H 2 O 2 for
different cell types. Similarly, different concentrations of DTBP
might be required to optimize the maintenance of cellular protrusions for different cell types. Trials should be carried out with a cell
membrane marker (e.g., wheat germ agglutinin tetramethylrhodamine conjugate (WGA-Rhod, 1:200 in PBS) in Ibidi μ-dishes
(Ibidi)) and check for cellular protrusion stability under a fluorescence microscope [26]. The cells should be washed twice with PBS
after fixation and labeled for 10–20 min in the dark at room
temperature with WGA-Rhod.
3.5 Laser Capture
Microdissection (LCM)
LCM parameters have to be set up to improve the isolation of
different cellular protrusions from the cells of interest. Here are
some key steps to take into account when using the LCM.
1. Mount the microdissection chamber dish onto the stage of the
LCM microscope and open the MMI Cell Tool program.
32
Ana Gordon and Karine Gousset
As previously stated, cellular protrusions are fragile and must be
fixed prior to LCM isolation [26, 27]. However, while a strong
fixative like glutaraldehyde is ideal for maintaining the integrity of
cellular protrusions, it is irreversible and does not allow for efficient
protein extraction post cell lysis (Fig. 1a, b). On the other hand, we
have recently demonstrated that using a cleavable crosslinker like
DTBP not only preserves cellular protrusions [26] but it also allows
for a greatly improved protein recovery after cell lysis (Fig. 1c).
Overall, the use of DTBP along with the alternate protein extraction protocol we have developed [26] allows for the reduction of
the overall number of cellular protrusions needed to be isolated by
LCM, as it greatly increases the number of spectra, number of
proteins, and number of total unique proteins identified by MS
compared to glutaraldehyde-fixed samples (Fig. 2).
1. To increase the number of cellular protrusions, a concentration
of 100 μM of H 2 O 2 in warm CAD media is added to the cells
for 5 min (see Note 10).
2. Add 100 μL for 500 μL total volume or 200 μL for 1 mL total
volume of the fixation solution 1 (4% PFA solution) directly to
the cells in their culture media and wait for 5 min.
3. Aspirate and add 1 mL of fixation solution 1 for 15 min (see
Note 11). Change fixative solutions by tilting the dish, allowing and carefully aspirating the solution near the wall, without
disturbing cells, or they might detach and cellular protrusions
could break. Take the same considerations for steps 4 and 5.
4. Aspirate and add 1 mL of fixation solution 2 (5 mM DTBP
solution) for 15 min.
5. Aspirate and add 1 mL of filtered PBS (see Note 12).
We recommend preestablishing the best conditions (time and
concentration) for induction of cellular protrusions with H 2 O 2 for
different cell types. Similarly, different concentrations of DTBP
might be required to optimize the maintenance of cellular protrusions for different cell types. Trials should be carried out with a cell
membrane marker (e.g., wheat germ agglutinin tetramethylrhodamine conjugate (WGA-Rhod, 1:200 in PBS) in Ibidi μ-dishes
(Ibidi)) and check for cellular protrusion stability under a fluorescence microscope [26]. The cells should be washed twice with PBS
after fixation and labeled for 10–20 min in the dark at room
temperature with WGA-Rhod.
3.5 Laser Capture
Microdissection (LCM)
LCM parameters have to be set up to improve the isolation of
different cellular protrusions from the cells of interest. Here are
some key steps to take into account when using the LCM.
1. Mount the microdissection chamber dish onto the stage of the
LCM microscope and open the MMI Cell Tool program.
32
Ana Gordon and Karine Gousset