Patient-derived GBM cells (U3034MG, U3088MG, and U3031MG)
or glioma cells (GCs) were grown in 6-well plates (50,000 cells/well),
coated with poly-l -ornithine (15 μg/mL) and laminin
(10 μg/mL), and grown to a confluency of 80%. Cells were expanded
in medium containing neurobasal, DMEM:F12 glutamax, media supplements,
and EGF and FGF (10 μg/mL, 1:1000) every 72 h. The plates were
incubated with p-HTMI (stock solution of 1 mg/mL in deionized water,
diluted 1:500) for 10 min, then incubated with Accutase for 7 min,
and resuspended in cell culture medium. Cells were spun down at 1500
rpm for 5 min. Further, they were incubated with binding buffer (BSA,
0.5 M EDTA, and PBS) (100 μL/sample) and CD133-APC (130-090-826
Mitenyi Biotec) (10 μL/sample), incubated for 10 min at 4 °C,
and washed with PBS prior to FACS analysis. Cells were incubated with
CD44 (45-0441 eBioscience) diluted in binding buffer and used at a
concentration of 1:10,000. The same antibody conditions as CD133 were
used for CD44 i.e. 100 μL of binding buffer, incubated for 10
min at 4 °C, and washed with PBS prior to FACS analysis. Cells
were also double stained for p-HTMI and CD271 (560834 BD Pharmingen),
5 μL/sample with the same antibody conditions as CD133. U-87MG
cells were expanded as previously described26 (link) and incubated with the same conditions of p-HTMI and CD133 and CD44
as described for GCs. To verify cell necrosis, lysis buffer was added
with 5 μL of PI per sample, and for cell apoptosis, cells were
incubated with 100 μL of 1X annexin V binding buffer and 5 μL
of APC annexin V per sample (561012 BD Pharmingen) and incubated for
15 min. The analysis was carried out on an FACS LSRII flow cytometer
equipped with FACSDiva software. Patient tissue collection and use
were in accordance with ethical permit EPN Uppsala 2007/353 and its
addendum Oct. 28, 2013.
or glioma cells (GCs) were grown in 6-well plates (50,000 cells/well),
coated with poly-
(10 μg/mL), and grown to a confluency of 80%. Cells were expanded
in medium containing neurobasal, DMEM:F12 glutamax, media supplements,
and EGF and FGF (10 μg/mL, 1:1000) every 72 h. The plates were
incubated with p-HTMI (stock solution of 1 mg/mL in deionized water,
diluted 1:500) for 10 min, then incubated with Accutase for 7 min,
and resuspended in cell culture medium. Cells were spun down at 1500
rpm for 5 min. Further, they were incubated with binding buffer (BSA,
0.5 M EDTA, and PBS) (100 μL/sample) and CD133-APC (130-090-826
Mitenyi Biotec) (10 μL/sample), incubated for 10 min at 4 °C,
and washed with PBS prior to FACS analysis. Cells were incubated with
CD44 (45-0441 eBioscience) diluted in binding buffer and used at a
concentration of 1:10,000. The same antibody conditions as CD133 were
used for CD44 i.e. 100 μL of binding buffer, incubated for 10
min at 4 °C, and washed with PBS prior to FACS analysis. Cells
were also double stained for p-HTMI and CD271 (560834 BD Pharmingen),
5 μL/sample with the same antibody conditions as CD133. U-87MG
cells were expanded as previously described26 (link) and incubated with the same conditions of p-HTMI and CD133 and CD44
as described for GCs. To verify cell necrosis, lysis buffer was added
with 5 μL of PI per sample, and for cell apoptosis, cells were
incubated with 100 μL of 1X annexin V binding buffer and 5 μL
of APC annexin V per sample (561012 BD Pharmingen) and incubated for
15 min. The analysis was carried out on an FACS LSRII flow cytometer
equipped with FACSDiva software. Patient tissue collection and use
were in accordance with ethical permit EPN Uppsala 2007/353 and its
addendum Oct. 28, 2013.
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