Biomedical Engineering Reference
In-Depth Information
Fig. 1
Phase-contrast photomicrograph of MCF10A spheroid colonies that were selected for the ability of
anchorage-independent growth (
a
) from MCF10A retroviral-infected cells in a soft agar screen (
b
)
4. After 24 h, gently overlay the agar with 15 mL 10A-medium,
supplemented with 1
g/mL puromycin. Twice a week, add
5 mL fresh medium (containing puromycin) to each dish
(
see
Note 16
).
5. Regularly inspect the dishes; after approximately 10 days, small
specks, indicating developing colonies, may be observed. After
3-4 weeks, colonies would have grown suffi ciently to be clearly
visible to the naked eye (Fig.
1
).
6. When colony size is satisfactory, move individual colonies to
individual wells in a 24-well plate.
7. Set up 24-well plates that contain 1 mL 10A-medium contain-
ing puromycin in each well. Label clearly, identifying the origi-
nating dish.
8. Using a sterile 1 mL pipette tip, while depressing the plunger
of the pipette, position the tip very close to the colony. While
slowly releasing the plunger, move the colony into the tip.
With some practice, this can be achieved with aspiration of
minimal amounts of liquid and agar.
9. Visually examine the contents of the tip to ascertain that a sin-
gle colony has been picked up. If multiple colonies are
observed, discard the colonies. Only proceed when single col-
onies are observed.
10. Expel the contents of the tip into one of the wells of the pre-
pared 24-well plate. Rinse the inside of the tip by pipetting up
and down a few times. This will also help to break up the
spheroid of cells. Incubate in TC incubator.
ΚΌ
