Biomedical Engineering Reference
In-Depth Information
cytospheres that can then be further studied. The interaction of
such combinational PPIs may lead to increasing the understand-
ing of cellular transformation with respect to various aspects of
cancer. In Part I (Subheading
3.1
), we describe a method of intro-
ducing secretory proteins carried by a retroviral library into
MCF10A cells with the goal to achieve multiple infections within
individual cells and to identify novel protein-protein combina-
tions that bring about cellular differentiation with the aim of fur-
ther understanding the development of cancer. In Part II
(Subheading
3.2
) and Part III (Subheading
3.3
), we describe sev-
eral means of evaluating the protein combinations by intracellular
as well as extracellular ways using the anchorage-independent
assay (soft agar assay) [
9
].
2
Materials
1. Amphotropic Phoenix packaging cell line (AMPHO cells;
Nolan Laboratory, Stanford University) [
10
,
11
], MCF10A
(ATCC), and pBABE human cDNA library [
8
].
2. Dulbecco's modifi ed Eagle's medium (DMEM) (Invitrogen)
supplemented with 10 % Fetal Bovine Serum (FBS; HyClone).
3. Phosphate buffered saline (PBS; Sigma): 137 mM NaCl,
2.7 mM KCl, 4.2 mM Na
2
HPO
4
, 1.4 mM KH
2
PO
4
, pH 7.4.
4. 10A-medium: Dulbecco's modifi ed Eagle's medium supple-
mented with 5 % horse serum (Invitrogen), 20 ng/mL epider-
mal growth factor (PeproTech), 0.5 mg/mL hydrocortisone
(Sigma), 100 ng/mL cholera toxin (Sigma), 10
2.1 Part I
2.1.1 Creation of Viral
Library and Infection
of MCF10A
ʼ
g/mL insulin
(Sigma).
5. 0.25 % Trypsin-Versene (HyClone).
6. Effectene Transfection Reagent (Qiagen).
7. Hemocytometer.
8. 5 mg/mL Polybrene (Hexadimethrine bromide, Sigma).
9. Puromycin (Sigma) (
see
Note 1
).
10. 37 °C, 5 % CO
2
cell culture incubator.
11. 32 °C, 5 % CO
2
cell culture incubator.
12. 0.45
M cellulose acetate (CA) vacuum fi lter units (Corning).
13. Sterile 15 mL polystyrene tubes with screw closure; 50 mL
sterile polypropylene centrifuge tube with PP screw closure
(Nalgene).
14. High-speed centrifuge, capable of speeds >32,000 rcf, and
50 mL tube rotors.
ʼ
