Muse autophagy lc3 antibody based kit

Autophagy assays were performed using Muse™ Cell Analyzer from Millipore (Hayward, CA, USA) following the manufacturer’s instructions. Following treatment of MGH-U3 and T24 cells with heparinase inhibiter RK-682 (Cayman Chemical Company, Ann Arbor, MI, USA), the treated cells were washed with PBS buffer. The autophagy assays were analyzed using the Muse™ Autophagy LC3-antibody based kit (Millipore) according to the manufacturer’s protocol.

The K562 and U-251 cells were seeded in 96-well plates (Nunc) and incubated at 37 °C for 24 h. The density of the cells was 20,000 cells per well (for the 24 h assay) and 10,000 per well (for the 48 h assay). Next, the medium was changed for freshly prepared solutions of the tested compounds (BS1, BS4) at 0.5 µM (for K562) and 4 µM (for U-251) concentrations, and the cells were incubated for 24 h or 48 h, respectively. Additionally, the cells were treated with imatinib (0.4 µM for K562 and 25 µM for U-251) as the positive control. After treatment, the assays were performed using a Muse™ Autophagy LC3-antibody based kit (Millipore) according to the manufacturer’s instructions. Briefly, the cells were collected, washed with cold HPBS and centrifuged at 300 g for 5 min. Then, the cells were resuspended in a mixture of 95 µL 1X Autophagy Reagent B and 5 μL Anti-LC3 Alexa Fluor^® 555 antibody. The samples were incubated on ice for 30 min in the dark. After incubation, the cells were centrifuged and resuspended in 200 μL of a 1X Assay Buffer. Then, the samples were directly processed to analyze the autophagy induction using a Muse Cell Analyzer. The autophagy induction ratio was calculated on the basis of the ratio between the target sample fluorescence versus the control sample. The experiments were performed at least three times.

PANC-1 and AsPC-1 cells were seeded in 96-well plates (Nunc) at a density of 20,000 cells/well (for 24 h assay) and 10,000 cells/well (for 48 h assay) and incubated at 37 °C for 24 hours. Then, the medium was removed and freshly prepared solutions of glycofullerenes at 1 mg/mL concentration were added and the cells were incubated for 24 or 48 hours. As positive control, cells were treated with Imatinib (25 µM) for 24 hours. After treatment, assays were performed using a MuseAutophagy LC3-antibody based kit (Millipore) according to the manufacturer’s instructions. Briefly, cells were collected, washed with cold HBSS and centrifuged at 300 g for 5 min. Afterwards, cells were resuspended in 95 μL of 1X Autophagy Reagent B with 5 μL of Anti-LC3 Alexa Fluor555 antibody and incubated for 30 minutes on ice in the dark. After this time, the cells were centrifuged and resuspended in 200 μL of 1X Assay Buffer. Then, the cells immediately were processed for autophagy induction analysis using a Muse Cell Analyzer (Millipore). Autophagy induction ratio were calculated based on the ratio between the target sample fluorescence versus the control sample. The experiments were performed at least three times.