Biology Reference
In-Depth Information
transfected cells can be stained 10-14 day after transfection.
For weaker cooperating oncogenes, such as Ad E1B, cells can
be stained 15-20 day after transfection.
3.3 Isolation and
Cultivation of Human
Amniotic Fluid Cells
(AFC)
Amniotic fl uid is a rich source for a mixture of different human cell
types. Out of this unspecifi c cell pool it is easy to isolate plastic
adherent cells, which can be further cultivated and propagated.
Here we show an easy method to isolate and cultivate AFCs for the
study of viral oncogenic transformation.
1. 10-15 mL freshly isolated amniotic fl uid was centrifuged for
5 min at 800 ×
g
and RT.
2. Discard the supernatant and resuspend the pellet in 2-3 mL
BIO-AMF-2 medium, and plate the whole volume into one
well of a 6-well tissue culture plate.
3. 7 days post plating aspirate the medium and feed the plastic
adherent cells with 2-3 mL of fresh BIO-AMF-2 medium.
4. If cells are 70-80 % confl uent, trypsinize them and passage
them to a 10-cm dish containing 8-10 mL Ham's F10 medium
supplemented with 10 % FCS, 1 % Penicillin/Streptomycin
and 1 % Ultroser™ G.
5. Passage and cultivate cells until they have grown to an
appropriate number for your experiments.
3.4 Transduction
of AF Cells
To study the growth stimulating and transforming functions of
human Ad
E1
and
E4
oncogenes, lentiviral vector constructs
expressing the individual (viral) gene products (e.g., LeGO vector
derivatives encoding E1A-12/13S, E1B-55 kDa, E1B19kDa,
E4orf3, or E4orf6) or combinations thereof are transferred to
primary human AF cells (
see
Note 7
). We routinely transduce AF
cells using a modifi ed protocol of Weber et al., 2008 [
12
]. Make
sure to prepare duplicate or triplicate samples.
1. Seed 3 × 10
4
AF cells per well in 500
μ
L medium in a 24-well
plate.
2. Wait 2-5 h for the cells to attach.
3. Replace medium with 500
μ
L of medium containing 8
μ
g/mL
Polybrene.
4. Add viral particle containing supernatant to the cells according
to an MOI of 0.3 particles per cell.
5. Centrifuge the plate for 1 h, 1,000 ×
g
at RT.
6. Change medium the next day. Use 1 mL per well (without
Polybrene).
7. Cultivate cells for another 3 days.
8. Transfer cells to 10-cm dishes.
