6. Keep Collagen Rat tail type I on ice anytime it is not stored at
4
C to avoid polymerization. Different batches may have
different pH and concentration. Anytime a new batch is used,
calculate volume needed to reach 4 mg/mL concentration and
verify that the gel mix solution becomes salmon colored once
properly mixed.
7. Matrigel concentration varies from one batch to another, consider adjusting all volumes of the ECM mixture when changing
batch. The final concentration should be 2 mg/mL.
8. To image thick samples such as 3D embedded spheroids with
high magnification and high numerical aperture objectives
(e.g., 40Â NA 0.75) it is important to use bottom glass dishes
with glass thickness #0 (0.085–0.115 mm) for optimal microscopy imaging.
9. The choice of the objective will depend on the quality of the
fluorescent signal and on the purpose of the assay. If the signal
to noise ratio of the fluorescent signal is high enough and the
migration features are easy to describe (e.g., cell strands, chains,
or clusters) then the positioning of cells in strand can be
assessed with a 10Â objective. To assess more precisely cellular
or subcellular events (e.g., cell–cell interactions, actin dynamic)
or if the fluorescent signal to noise ratio is deem, the use of
20–40Â with high numerical aperture (e.g., 20Â, NA 0.75;
40Â, NA 0.75) might be required.
10. During gel mixture preparation, slowly pipette the highly viscous collagen and Matrigel solution, allowing it to flow into
the 1 mL pipette tip after fully releasing the plunger button.
While dispensing, do not immerge the tip into the solution
(causing bubbles). Instead, pipette viscous reagents to the
sidewalls of the tube allowing the solution to flow to the
bottom of the tube. Avoiding bubbles is critical in this step.
We emphasize the importance of preventing drops containing
air bubbles. Indeed, they easily detach from the glass bottom
and reduce the quality of the acquisitions.
11. This step should be performed rapidly to avoid the polymerization of the first drops at room temperature while dispensing
new drops. Rapid deposition will also prevent the embedded
spheroids from precipitating into the drop, reaching the glass
bottom of the plate. This could result in a 2D migration of the
spheroids within the drop. To overcome this problem, we
suggest keeping the plate on ice while seeding the drops.
Alternatively, less drops can be plated for each experiment
using 6-well glass-bottom plates or single glass-bottom dishes.
12. Do not leave the ECM drops without media more than 40 min
in total (including the step of plate inversion and the following
20-min incubation) to prevent drying.
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4
C to avoid polymerization. Different batches may have
different pH and concentration. Anytime a new batch is used,
calculate volume needed to reach 4 mg/mL concentration and
verify that the gel mix solution becomes salmon colored once
properly mixed.
7. Matrigel concentration varies from one batch to another, consider adjusting all volumes of the ECM mixture when changing
batch. The final concentration should be 2 mg/mL.
8. To image thick samples such as 3D embedded spheroids with
high magnification and high numerical aperture objectives
(e.g., 40Â NA 0.75) it is important to use bottom glass dishes
with glass thickness #0 (0.085–0.115 mm) for optimal microscopy imaging.
9. The choice of the objective will depend on the quality of the
fluorescent signal and on the purpose of the assay. If the signal
to noise ratio of the fluorescent signal is high enough and the
migration features are easy to describe (e.g., cell strands, chains,
or clusters) then the positioning of cells in strand can be
assessed with a 10Â objective. To assess more precisely cellular
or subcellular events (e.g., cell–cell interactions, actin dynamic)
or if the fluorescent signal to noise ratio is deem, the use of
20–40Â with high numerical aperture (e.g., 20Â, NA 0.75;
40Â, NA 0.75) might be required.
10. During gel mixture preparation, slowly pipette the highly viscous collagen and Matrigel solution, allowing it to flow into
the 1 mL pipette tip after fully releasing the plunger button.
While dispensing, do not immerge the tip into the solution
(causing bubbles). Instead, pipette viscous reagents to the
sidewalls of the tube allowing the solution to flow to the
bottom of the tube. Avoiding bubbles is critical in this step.
We emphasize the importance of preventing drops containing
air bubbles. Indeed, they easily detach from the glass bottom
and reduce the quality of the acquisitions.
11. This step should be performed rapidly to avoid the polymerization of the first drops at room temperature while dispensing
new drops. Rapid deposition will also prevent the embedded
spheroids from precipitating into the drop, reaching the glass
bottom of the plate. This could result in a 2D migration of the
spheroids within the drop. To overcome this problem, we
suggest keeping the plate on ice while seeding the drops.
Alternatively, less drops can be plated for each experiment
using 6-well glass-bottom plates or single glass-bottom dishes.
12. Do not leave the ECM drops without media more than 40 min
in total (including the step of plate inversion and the following
20-min incubation) to prevent drying.
254
Sefora Conti et al.