Axio plan 2 uorescence microscope

Senescence was determined either by ow cytometry or immunostaining as two independent methods.
For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).

Senescence was determined either by ow cytometry or immunostaining as two independent methods. For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).