Acyclovir (topical, oral and parentral }

Procedure

1. View cultures using an inverted microscope to assess the degree of
confluency and confirm the absence of bacterial and fungal contaminants.

2. Remove spent medium.

3. Wash the cell monolayer with PBS without Ca2+/Mg2+ using a volume
equivalent to half the volume of culture medium. Repeat this wash step if the
cells are known to adhere strongly.

4. Pipette trypsin/EDTA onto the washed cell monolayer using 1ml per 25cm2
of surface area. Rotate flask to cover the monolayer with trypsin. Decant the
excess trypsin.

5. Return flask to the incubator and leave for 2-10 minutes.

6. Examine the cells using an inverted microscope to ensure that all the cells
are detached and floating. The side of the flasks may be gently tapped to
release any remaining attached cells.

7. Resuspend the cells in a small volume of fresh serum-containing medium
to inactivate the trypsin. Remove 100-200µl and perform a cell count (see
Protocol 6 - Cell Quantification, p.50). In the case of cells cultured in serum-
free media, use a trypsin inhibitor e.g. soyabean trypsin inhibitor to inactivate
the trypsin.

8. Transfer the required number of cells to a new labelled flask containing pre-
warmed medium (refer to the appropriate ECACC Cell Line Data Sheet for the
required seeding density).

9. Incubate as appropriate for the cell line.

10. Repeat this process as demanded by the growth characteristics of the
cell line.