Volocity 6.3 image analysis software

Western blots were quantified by densitometry using ImageJ (https://imagej.nih.gov/ij/). Immunofluorescence-based mitophagy assay quantitation was performed manually using NIS-Elements software (Nikon) as we previously described [10 (link)] or by Volocity 6.3 Image Analysis Software (PerkinElmer). Within each experiment, analysis was performed on at least 3 fields or 10 fields (H9c2 cells) of view (typically >50 cells per experiment) for all conditions tested. Data were not collected with the counter blinded to the condition, except for Fig. 1A. In Fig. 1A, a threshold of 3 or more red-alone puncta per cell was applied to the data to determine the number of cells undergoing mitophagy.

Data were quantified with Volocity 6.3 Image Analysis Software (PerkinElmer) using algorithms developed to analyze object overlap and count individual structures. For all analyses, we obtained images using uniform random sampling by an experimenter blind to all conditions. All images in each experimental group were processed as a batch using identical protocols. Images were first filtered to suppress noise (3x3 median filter) and a red/green intensity ratio channel was calculated. Similar analysis protocols were used for all eye regions. 1) The tissue was segmented using a fixed intensity threshold on the DAPI or green channel, followed by a fill holes operation and minimum size criterion. 2) To assess autophagy/mitophagy, objects were found in the red channel using mean intensity +n standard deviations (3 standard deviations for all tissues except the cornea, where the threshold was set at 2 standard deviations). Touching objects were separated using a guide size of 0.1 µm². These objects were filtered according to a minimum red/green ratio value: 0.7 for all tissues except lens (1.0) and ciliary body (3.0). Differences in threshold values reflect the fact that expression levels and imaging settings were different between these eye regions. Finally, objects not overlapping with the tissue were excluded.

To assess neutrophil migration in response to chemokines, Millicell EZ SLIDE 8-well glass chambers (Millipore, Cat #: PEZGS0816) were first incubated with 200 µl of 10 µg/ml recombinant mouse ICAM-1 plus 200 ng/ml of CCL4, CCL5, or CXCL12 overnight at 4 °C. On the 2nd day, both neutrophils and TGFβ1-expressing neutrophils (TNs) were freshly isolated from BM of 2- and 22-mon-old C57 mice using a Neutrophil Isolation Kit, and 1 × 10⁴ isolated cells were resuspended in 200 μl of L-15 medium (Gibco, Cat #: 11415114) and added into each glass chamber. Video recording of cell migration in a velocity assay was performed after incubation at 37 °C for 10 min. The migration video was recorded using a ×20 objective lens in a Nikon TE2000-U microscope, and image processing and velocity assay were performed using Volocity 6.3 Image Analysis Software by PerkinElmer, Inc. (PerkinElmer, Waltham, MA).