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We are experiencing some difficulty in analysing and sorting from a
population of transfected HEK cells. In particular, we are having the
following problems:
1. We have checked indo-1 loading by bulk fluorimetry, and have obtained
reasonable Fmin and Fmax. On our Vantage, this comes out as a very poor
signal, making ratio measurements difficult. We see other suspension
cultures, giving good data.
2. We see fairly poor scatter properties, but if we use propidium
iodide, viability seems to be reasonable.
We want to sort on the basis of calcium mobilisation, with the idea of
producing a "super-responding" population for out ligand.
Our questions are:
1. Does anyone have ideas on how to produce good quality single cell
suspensions from monolayer cultures, particularly with respect to HEK cells.
2. Is it sensible to try to produce this "super-responding" population
at all? When we tried to do the same on the basis of surface marker
expression, we saw that the original phenotype slowly returned over the
course of about 6 passages.
3. Would we be better off trying to dilution clone our cells?
>From a temporarily depressed flow lab
Graham
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