2 Materials
2.1 Cell Culture
Equipment
1. Cell incubator allowing CO 2 and temperature control.
2. Laminar flow hood for cell culture.
2.2 Cell Line
Immortalized human breast epithelial cell line MCF10A
[11]. Other cell lines may be used (see Note 1).
2.3 Cell Culture
Medium
1. Culture medium: basal medium DMEM/F12 (1:1) (1Â)
(31330–038 Gibco or any equivalent reference from other
suppliers).
2. Horse serum (Merck H1270 or any equivalent reference from
other suppliers). Aliquots from the same lot are conserved at
À20
C.
3. Stock solution of cholera toxin (Merck C8052 or any equivalent reference from other suppliers). 0.5 mg are re-suspended
in cell culture grade water at a final concentration of 1 mg/mL
and filtered using a 0.2 μm membrane. Aliquots are stored at
4
C (do not freeze).
4. Stock solution of insulin (Merck I9278, 1000Â solution or any
equivalent reference from other suppliers). The stock solution
is stored at 4
C.
5. Stock solution of hydrocortisone (Merck H0888 or any equivalent reference from other suppliers). 1 g re-suspended in
absolute ethanol at a final concentration of 1 mg/mL. Aliquots
are stored at À20
C.
6. Stock solution of Penicillin/Streptomycin (100Â), stored at
4
C.
7. Stock solution of L-Glutamine (100Â), stored at À20
C.
8. Stock solution of 0.1% bovine gelatine in PBS.
9. Accutase solution (1Â solution).
10. Stock solution of human recombinant epidermal growth factor
(hEGF) (StemCell Technologies or any equivalent reference
from other suppliers) at 1 mg/mL prepared following providers’ recommendation and stored at À20
C.
11. Stock solution of human recombinant Interleukin 6 (hIL6)
(StemCell Technologies or any equivalent reference from
other suppliers) at 10 μg/mL prepared following providers’
recommendation and stored at À20
C.
12. Stock solution of human recombinant Transforming Growth
Factor beta (hTGFβ) (StemCell Technologies or any equivalent reference from other suppliers) at 20 μg/mL prepared
following providers’ recommendation and stored at À20
C.
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
343
2.1 Cell Culture
Equipment
1. Cell incubator allowing CO 2 and temperature control.
2. Laminar flow hood for cell culture.
2.2 Cell Line
Immortalized human breast epithelial cell line MCF10A
[11]. Other cell lines may be used (see Note 1).
2.3 Cell Culture
Medium
1. Culture medium: basal medium DMEM/F12 (1:1) (1Â)
(31330–038 Gibco or any equivalent reference from other
suppliers).
2. Horse serum (Merck H1270 or any equivalent reference from
other suppliers). Aliquots from the same lot are conserved at
À20
C.
3. Stock solution of cholera toxin (Merck C8052 or any equivalent reference from other suppliers). 0.5 mg are re-suspended
in cell culture grade water at a final concentration of 1 mg/mL
and filtered using a 0.2 μm membrane. Aliquots are stored at
4
C (do not freeze).
4. Stock solution of insulin (Merck I9278, 1000Â solution or any
equivalent reference from other suppliers). The stock solution
is stored at 4
C.
5. Stock solution of hydrocortisone (Merck H0888 or any equivalent reference from other suppliers). 1 g re-suspended in
absolute ethanol at a final concentration of 1 mg/mL. Aliquots
are stored at À20
C.
6. Stock solution of Penicillin/Streptomycin (100Â), stored at
4
C.
7. Stock solution of L-Glutamine (100Â), stored at À20
C.
8. Stock solution of 0.1% bovine gelatine in PBS.
9. Accutase solution (1Â solution).
10. Stock solution of human recombinant epidermal growth factor
(hEGF) (StemCell Technologies or any equivalent reference
from other suppliers) at 1 mg/mL prepared following providers’ recommendation and stored at À20
C.
11. Stock solution of human recombinant Interleukin 6 (hIL6)
(StemCell Technologies or any equivalent reference from
other suppliers) at 10 μg/mL prepared following providers’
recommendation and stored at À20
C.
12. Stock solution of human recombinant Transforming Growth
Factor beta (hTGFβ) (StemCell Technologies or any equivalent reference from other suppliers) at 20 μg/mL prepared
following providers’ recommendation and stored at À20
C.
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
343