Click it tunel alexa fluor imaging assay

A 5 m in cytospin cycle at 500 rpm was used to attach MCL cells to glass slides. Cells were stained with the Click-iT TUNEL Alexa Fluor Imaging assay (Invitrogen C10245) per manufacturer’s instructions and imaged on an Olympus BX60 upright fluorescence microscope.

Cells were grown on glass coverslips, treated for 24 h and then washed with PBS and fixed in 4% formaldehyde for 15 min at room temperature. Fixed cells were washed with PBS and then soaked for 20 min with 0.25% of Triton X-100 in PBS. After two washes in deionized water, they were stained using the Click-iT^® TUNEL Alexa Fluor^® Imaging Assay (Invitrogen) according to the manufacturer's protocol. Co-staining with Hoechst33342 was performed to analyze the nuclear morphology of the cells after the treatment. Cell nuclei were observed and imaged under an inverted fluorescence microscope (200X magnification).

Analysis of apoptosis was performed using TUNEL kit (Click-iT TUNEL Alexa Fluor Imaging Assay, Invitrogen) following the manufacturer’s protocol. Briefly, cells were plated at 2.5 × 10⁵ cells/six-well plate. After adhesion, the cells were treated with cisplatin (50 μM) alone or combined with either 5 μg/ml of PFOTE or OTE for 24 h before analysis. After fixation and permeabilization, cells were stained with TUNEL reaction mixture containing FITC-dUTP. Apoptotic nuclei (green) were observed under an Olympus inverted fluorescence microscope IX71S1F-3 (× 20).

For the detection of DNA fragmentation, the Click-iT^® TUNEL Alexa Fluor^® Imaging Assay (Invitrogen) was used according to the manufacturer's instructions. Briefly, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized in 0.5% Triton^™ X-100 in PBS for 20 min at room temperature. The cells were then incubated for 60 min at 37°C in terminal deoxynucleotidyl transferase enzyme reaction mixture. The cells were washed twice with 1X PBST for 2 min each and then incubated for 30 min at room temperature with Click-iT^® reaction mixture. Cell nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich) for 15 min at room temperature and images were taken under a fluorescence microscope.

For phase-contrast microscopy and for immunostaining, intact or partially squashed testes from 2 to 4 days old wild-type and mutant flies were processed as described earlier [55 ]. DAPI (1 µg ml⁻¹) was used for DNA staining and Texas Red-X Phalloidin (Invitrogen) was used in 1 : 250 dilution for actin visualization. Primary antibodies used: rabbit anti-Lasp, 1 : 200 (gift from Anne Ephrussi); mouse anti-Calnexin99A, 1 : 100 (gift from Sean Munro); rabbit anti-cleaved-Caspase3, 1 : 200 (clone 5A1E, Cell Signalling). Alexa Fluor 488 conjugated anti-rabbit secondary antibody was from Invitrogen. TUNEL labelling of the testis was carried out using Click-iT TUNEL Alexa Fluor Imaging Assay (Invitrogen) as described in Kibanov et al. [56 (link)]. The samples were mounted in Fluoromount (Southern Biotech) and imaging was done with an Olympus BX51 fluorescent microscope or with an Olympus FV 1000 confocal microscope. Signal quantification for RFP-PH-FAPP and PLCδ-PH-GFP was done on images collected using the same exposure time for different genotypes. Membrane to cytoplasmic fluorescent signal ratios were calculated from the measurements of mean pixel intensities within equal areas of membrane versus cytoplasm (n = 20). Measurements were done using ImageJ software.