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Cells were harvested after one passage to generate enough material for IP assays and one or two passages for protein isolation.

After growing to confluence, cells were subjected to two passages to generate pure astrocytic cultures.

These primary spheres were passaged to generate the cells to be used in experiments.

Primary mammo-spheres can be further passaged to generate secondary mammospheres.

We first deleted the speE gene of C. glutamicum ATCC 13032 (ΔargFΔproB), then evolved by a growth-based selection process for 70 passages to generate C. glutamicum ΔAPE6937, and finally deleted the argR gene of the evolved strain to obtain C. glutamicum ΔAPE6937R42.

Since CD9-transfected clones eventually died, we subcloned the pooled population (at passage 2) to generate more CD9-transfected PC-3 clones.

We next analyzed the propensity of manually passaged hESC lines to generate CD34+ and CD34+CD45+ using a complementary differentiation system.

Primary MEFs (passage 4) were used to generate the HA-eIF4E-expressing MEFs as described above.

The resulting cell lysates were used to generate passage 1 stocks by re-infecting RD cells.

Each isolate was given a second passage in Vero cells to generate a working stock and stored at –80°C.