Annexin 5 fitc staining kit

The glioma cells were plated in six-well plates and incubated overnight to adhere. Then the media was changed to complete culture media with temozolomide. The cells were treated for 72 h then collected by trypsinization and stained by Annexin V-FITC staining kit (Abcam). The cells were analyzed by flow cytometry and the FITC⁺ cells were determined as apoptotic. Representative gating strategy to determine apoptotic cells is shown in Supplementary Fig. 7d.

The externalisation of phosphatidylserine as the first symptom of apoptosis was determined using an Annexin V-FITC staining kit (Biovision, USA). In parallel, propidium iodide (PI) staining was conducted for cellular membrane integrity analysis. After 48 and 72 h continuous incubation with PTX alone or PTX-NCs, at doses of 12.5, 25, and 50 nM, the cells were collected by trypsinization centrifuged at 1000 rpm for 10 min, stained with the supplied fluorochromes and analysed using a LSRII BD cytometer equipped with the FACS Diva LSR II software (BD Dickinson, New York, NY, USA), the obtained data are expressed as cell percentages [30 (link)]. In parallel, the molecular features of apoptosis, after staining the cells with Annexin V and Hoechst 33,258, were visualised with a confocal microscope (Leica SP-8, Leica Microsystems, Wetzlar, Germany) with a 63×/1.40 objective (HC PL APO CS2, Leica Microsystems). Leica Application Suite X software (LAS X, Leica Microsystems, Germany) was used to obtain the images.