We have been trying to optimise the Pershouse, Sanford and Stubblefield
MMCT protocol for the last 8 months.
We have an ovarian cancer cell line which is either neomycin or hygromycin
tagged. We can make microcells abundantly from the neo resistant line but
we canÕt get doubly resistant hyg/neo lines postfusion. We canÕt detect a
synergistic effect between the 2 antibiotics
We use 250ug/ml PHAPin Ca/Mg free HBSS and attach at 37 C for 20min.
We can see the microcells firmly attached to the cells and the flask prior
to PEG fusion. We use 50% PEG1500 sterile, Ca free, in 75mM Hepes for 1min
(2ml per 25ml flask)at room temp for fusion.
We have done 20 experiments with no colonies out the other end --- are we
doing something glaringly wrong? Can anyone suggest where we should proceed
Please reply via Email.
Hani Gabra (Edinburgh, Scotland)