For experiments mimicking hypoxic conditions, cells were cultured with 100 μM deferoxamine mesylate (DFX) (Sigma-Aldrich), a hypoxia mimetic[21 (link),22 (link)]. A Silencer Select short interfering RNA (siRNA) against human HIF-2α (Dharmacon, Lafayette, CO) was delivered to cells using two different transfection systems: (1) Lipofectamine RNAiMAX as described by the manufacturer (Invitrogen); and (2) cationic amphiphilic micelles complexed with transfection lipids as described in a previous publication[23 (link)]. Silencer Select Negative Control siRNA (Invitrogen) or Luciferase siRNA (Invitrogen) was used as a control, nontargeting sequence. After 24 h, 1 mM temozolomide (Invitrogen) was added to the media. After 48 h, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Promega, Madison, WI) was performed to evaluate viability as a measure of chemotherapeutic response to a combined HIF-2α siRNA and temozolomide therapy.
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide assay
Manufactured by Promega
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay is a colorimetric assay used to measure cell viability and proliferation. It involves the conversion of the tetrazolium dye MTT into a purple-colored formazan product by metabolically active cells.
Automatically generated - may contain errors
2 protocols using 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide assay
1
Hypoxia Mimicry and HIF-2α Silencing Enhance Chemotherapy
2
Hypoxia Mimetic and HIF-2α Knockdown Enhance Temozolomide Sensitivity
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For experiments mimicking hypoxic conditions, cells were cultured with 100 μM deferoxamine mesylate (DFX) (Sigma-Aldrich), a hypoxia mimetic[21 (link),22 (link)]. A Silencer Select short interfering RNA (siRNA) against human HIF-2a (Dharmacon, Lafayette, CO) was delivered to cells using two different transfection systems: (1) Lipofectamine RNAiMAX as described by the manufacturer (Invitrogen); and (2) cationic amphiphilic micelles complexed with transfection lipids as described in a previous publication[23 (link)]. Silencer Select Negative Control siRNA (Invitrogen) or Luciferase siRNA (Invitrogen) was used as a control, nontargeting sequence. After 24 h, 1 mM temozolomide (Invitrogen) was added to the media. After 48 h, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Promega, Madison, WI) was performed to evaluate viability as a measure of chemotherapeutic response to a combined HIF-2a siRNA and temozolomide therapy.
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