5. CAFs culture medium, warmed at 37
C before use. Keep a
500 μL aliquot at 4
C.
6. 12-Well glass-bottomed cell culture plates, glass thickness #0
(0.085–0.115 mm) (MatTek) (see Note 8).
7. ECM mixture (see Table 2):
5ÂCol solution.
Matrigel. The aliquot should be thawed the day before the
experiment in a box of ice at 4
C.
FBS, filtered.
CAFs medium (see Note 3).
Rat tail collagen type I.
2.4 Image
Acquisition
and Analysis
1. Confocal microscope with and environmental chamber for
temperature and CO 2 control and a motorized stage for multiple XYZ positions.
Table 1
Cell suspension solution for hanging-drop culture
Mix 1:1 ratio
CAFs:CC
Mix 2:1:1 ratio
CAFs:CC 1 :CC 2
1.2% Methylcellulose solution
200 μL
200 μL
CAF suspension
(750,000 cells/mL)
83.5 μL
83.5 μL
Cancer cell suspension
(750,000 cells/mL)
83.5 μL
C C 1 41.75 μL
CC 2 41.75 μL
CAF culture medium
633 μL
633 μL
CC cancer cell, CC 1 cancer cell type 1, CC 2 cancer cell type 2
Table 2
Collagen I/Matrigel gel mixture
Volume (μL)
Matrigel
225
Rat tail collagen type I
421
5ÂCol
84
FBS
100
Culture medium
120
Pelleted spheroid (remaining volume)
50
246
Sefora Conti et al.