184
Established GBM cell lines (U87, 8MG) and primary cultures were
cultured in DMEM or DMEM/F12, respectively, supplemented
with 10% FBS and maintained at 37 °C in a 5% CO 2 -humidified
incubator. Established lines were purchased from ATCC and primary cells were generated in-house from fresh brain tumor tissue
as described previously by Renziehausen et al. [8].
1. Seed the cells in 96-well plates at 2 × 10
3
cells per well in their
respective culture medium supplemented with 2% FBS.
2. Treat 24 h post-plating cells with TMZ, PSC4, a physical mixture of TMZ and PSC4, or TMZ@PSC4 complex in culture
medium supplemented with 2% FBS.
3. To determine the IC 50 of native TMZ, treat cells with 2, 4, 8,
16, 32, 64, 128, 256, and 512 μM TMZ and analyze for proliferation using SRB assay 9  days posttreatment as previously
described [16].
4. For experiments comparing the efficacy of the PSC4 complex
with native TMZ, use doses just below the IC 50 of native TMZ
(5 μM and 10 μM for U87 and 8MG, respectively; 100 μM and
200 μM for the primary lines).
5. Calculate and use the equivalent equimolar concentrations of
the complex.
3.3 Cell Culture
3.4 In Vitro
Cytotoxicity:
Sulforhodamine B
(SRB) and Cell
Counting Kit 8 (CCK8)
Assays
Fig. 7 The degradation rate of TMZ and TMZ@PSC4 in mice plasma. TMZ@PSC4
is more stable 4 h post-dosage in comparison to TMZ
Antonis D. Tsiailanis et al.