Papain

Papain was from Roche (Mannheim, Germany). All other substances used were from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. The following primary antibodies were used: rabbit anti-Kir4.1 (1 : 200; Sigma-Aldrich), mouse anti-GFAP (1 : 200; G-A-5 clone, Sigma-Aldrich), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1 : 500, Wako, Neuss, Germany), goat anti-calretinin (1 : 500, Swant, Marly, Switzerland), mouse anti-calbindin (1 : 400, Swant), rabbit anti-PKCα (1 : 300, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-CRALBP (1 : 300, Santa Cruz Biotechnology) and mouse anti-glutamine synthetase (1 : 1000, Merck Millipore, Darmstadt, Germany). The following secondary antibodies were used: Cy5-conjugated donkey anti-goat, Cy3-conjugated donkey anti-rabbit, Cy2-conjugated donkey anti-mouse, Cy3-conjugated goat anti-rabbit and Cy2-conjugated goat anti-mouse. All secondary antibodies were applied in a 1 : 200 dilution and were obtained from Dianova (Hamburg, Germany). The apoptosis rate was detected using the in situ cell death detection kit, tetramethylrhodamine red (Roche).

Adult animals expressing the mito-Dendra2 tag were anesthetized with isoflurane and decapitated. Retinas were immediately explanted and dissected in 1X PBS on ice. Each retina was transferred to a 1.5ml Eppendorf low-adhesion tube filled with the 800μl digestion buffer (1:8:1 Cysteine/EDTA solution (2.5mM Cysteine, 0.5mM EDTA (ethylenediaminetetraacetic acid) in HBSS, 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in HBSS, and 10mg/ml Papain, Roche #10108014001), incubated at 37°C for 10 minutes, then centrifuged for 2.5 min at 240xg. Samples were transferred on ice, the supernatant was discarded and 800μl 1mM EDTA in HBSS + 2% (v/v) FBS added. After two washes with 1mM EDTA in HBSS + 2% (v/v) FBS, the digested tissue was triturated 10-15 times with a pulled glass pipette, then filtered through a 70 μm strainer. For the measurement of mitochondrial reactive oxygen species, 5 μM MitoSox (ThermoFisher, #M36008) in HBSS was added to the digested tissue and pipetted to break apart the tissue prior to a 10 minute incubation at 37°C, then the sample was centrifuged for 3 minutes at 304xg at 4°C. Samples were carefully washed two times with 1mM EDTA in HBSS + 2% (v/v) FBS, triturated 10-15 times with a pulled glass pipette, then filtered through a 70 μm strainer.

To investigate whether proteases are required to form extracellular traps in coelomocytes, protease inhibitor cocktail (cOmplete Protease Inhibitor Tablets, Roche) was used as a broad protease inhibitor (BROAD PROT). It inhibits serine, cysteine, and metalloproteases in bacterial, mammalian, yeast, and plant cell extracts (CO-RO, Roche Mannheim, Germany) [54 (link)]. Additionally, we used [phenylmethlysulfonyl] fluoride (PMSF, Roche, Mannheim, Germany) that inhibits mainly serine proteases (1 mM, SERINE PROT) [55 (link)]. One milliliter of BROAD PROT contains: 375 μg/ml pancreas extract; pronase, 0.375 μg/ml; thermolysin, 0.2 μg/ml; chymotrypsin, 0.37 μg/ml; trypsin, 0.5 μg/ml; papain, 250 μg/ml (Roche, Mannheim, Germany). 10 μl of inhibitors were added to the cells in vitro or earthworms (the in vivo setting) were injected with 20 μl.

All substances were purchased from Sigma‐Aldrich (Taufkirchen, Germany) unless stated otherwise. Papain was obtained from Roche (Mannheim, Germany). Chloromethyl‐tetramethyl‐rosamine (Mitotracker Orange), NP‐EGTA (o‐nitrophenyl EGTA), NPE‐ATP (P(3)‐[1‐(2‐nitrophenyl)]ethyl ester of ATP), and Fluo‐4 AM were from Molecular Probes (Life Technologies, Carlsbad, CA). For immunocyto‐ and histochemical staining, the following primary antibodies were used: rabbit anti‐Kir4.1 (1:200; Sigma‐Aldrich), mouse anti‐glial fibrillary acidic protein (GFAP; 1:200; G‐A‐5 clone, Sigma‐Aldrich), goat anti‐calretinin (1:500, Swant, Marly, Switzerland), mouse anti‐calbindin (1:400, Swant), rabbit anti‐PKCα (1:300, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti‐cellular retinaldehyde‐binding protein (CRALBP; 1:300, Santa Cruz), rabbit‐anti‐Iba1 (1:500, Wako Chemicals, Neuss, Germany), and mouse anti‐glutamine synthetase (1:1000, Merck Millipore, Darmstadt, Germany). As secondary antibodies, we used Cy5‐conjugated donkey anti‐goat, Cy3‐conjugated donkey anti‐rabbit, Cy2‐conjugated donkey anti‐mouse, Cy3‐conjugated goat anti‐rabbit, and Cy2‐conjugated goat anti‐mouse. All secondary antibodies were obtained from Dianova (Hamburg, Germany) and applied at 1:200 dilution. Apoptosis was detected by the in situ cell death detection kit (Roche, Mannheim, Germany).