The cell concentration and the viability were determined using a classic dye exclusion method (Trypan Blue).
After using a live/dead exclusion dye (Molecular Probes, Eugene, OR, USA) an Fc blocking step (anti-mouse CD16/32, TruStain fcX™, Biolegend, CA, USA) was used to reduce non-specific binding.
Hemacytometer counts using erythrosine B dye exclusion were used to assess cellular density and viability.
Cellular viability was examined by counting the viable cells using trypan blue dye exclusion, and cellular proliferation was measured using an MTS proliferation assay kit (Promega, Madison, WI, USA).
Additionally, post-thaw viability of samples were determined after a rapid thaw and 15 min incubation period using a fluorescence membrane dye exclusion assay.
To determine whether expression of the CTPsyn-YFP transgene had an adverse effect on cell survival, cells were assayed for viability using a trypan blue dye exclusion assay.
The cytotoxic effect of TMZ on malignant glioma cell lines was determined by using a trypan blue dye exclusion assay as described previously (Mukai et al, 2000).
The effect of CDKIs on cell viability was determined by using a trypan blue dye exclusion assay as described previously (Komata et al, 2000).
The effect of anti-PDGF neutralising antibody on cells was determined by using a trypan blue dye exclusion assay as described previously (Komata et al, 2000).
Cells were dissociated in 'initial' plating medium: Neurobasal medium (Gibco BRL, Life Technologies), 2%B277 supplement (Gibco BRL, Life Technologies), 10% foetal bovine serum, 0.5 mM glutamine, 25 μM glutamate and penicillin/streptomycin. Cell density and viability was assessed using a Trypan Blue dye exclusion assay, and the volume adjusted to achieve a density of 8×10 cells/ml.
