Mito id red detection kit

The cells were grown on cover slips in 24-well plates at 5000 cells per well and treated with temozolomide (50μM), doxycycline (50μM) or a combination of both drugs for 2 cycles at 72h each. For fluorescence staining of mitochondria the MITO-ID Red Detection Kit (Enzo) was used following manufacturer’s instructions.

HeLa cells stably expressing untagged Parkin WT, S65A or H302A were plated on glass coverslips and treated as described. Immunofluorescence was performed as described in 33 (link). Briefly, coverslips were washed twice with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde, 50 mM HEPES pH 7.0 for 10 min, washed twice with and then incubated for 10 min with DMEM, 10 mM HEPES pH 7.4. Cells were permeabilised by incubation with 0.2% Triton X-100 in PBS followed by two washes and blocking for 15 min at RT with PBS supplemented with 1% BSA (PBS/1% BSA). Cells were stained with the primary antibodies as follows: Parkin^{Phospho-Ser65} (1:500) antibody for 16 h at 4°C and total Parkin antibody (1:1,000) for 1 h at 37°C, followed by anti-mouse or anti-rabbit Alexa Fluor 405- or Alexa Fluor 488-conjugated secondary antibodies (Life Technologies). The mitochondria was stained using MITO-ID® Red detection kit (Enzo Life Sciences) at for 30 min at 37°C.
Immunofluorescently labelled cells were imaged using the Zeiss LSM 700 laser scanning confocal microscope with the Alpha Plan-Apochromat ×100/NA 1.46 objective (optical section thickness 0.7 μm). Parkin-Alexa 405 was excited with the 405 laser, P-Parkin-Alexa 488 was excited with the 488 laser, and Mitochondria MITO-ID® Red was excited with the 555 laser. All labels were excited independently to prevent cross-channel bleed through.

The hemocyte cell cultures were prepared according to Section 2.4. After 24 h of incubation, the cells were fixed in 4% paraformaldehyde (Sigma Aldrich, München, Germany; PFA) in phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100 (Sigma Aldrich, München, Germany) in PBS. Mitochondria were detected with a MITO-ID Red Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA). The cells were incubated for 30 min with Dual Detection Reagent and ActinGreen 488 ReadyProbes Reagent (Invitrogen, Thermofisher, Carlsbad, CA, USA) was used to label the actin fibres. The cell nuclei were stained with Hoechst (Enzo Life Sciences). Fluorescence signals were analysed by fluorescent microscopy using an Axio Vert.A1 fluorescence microscope (Carl Zeiss, Jena, Germany) with Axio Cam ICc 5 (Carl Zeiss, Jena, Germany).